Methods for production of oxygenated terpenes

ABSTRACT

The present disclosure relates to methods for producing oxygenated terpenoids, and preparation of compositions and formulations thereof. Polynucleotides, derivative enzymes, and host cells for use in such methods are also provided.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. patent application Ser. No.16/669,051, filed on Oct. 30, 2019, which is a divisional of U.S. patentapplication Ser. No. 15/505,022, filed Feb. 17, 2017, which is a UnitedStates National Stage Application of International Application No.PCT/US2015/046421, filed Aug. 21, 2015, which claims the benefit of, andpriority to, U.S. Provisional Application No. 62/040,284, filed Aug. 21,2014, each of which is incorporated herein by reference in its entirety.

FIELD OF THE INVENTION

The present disclosure relates to oxygenated sesquiterpenes (e.g.,nootkatone and/or nootkatol) and methods for their production and use.The disclosure also provides enzymes for the production of oxygenatedsesquiterpenes (e.g., nootkatone and/or nootkatol) and methods foridentifying, selecting, making and using these enzymes.

BACKGROUND OF THE INVENTION

The food and beverage industries as well as other industries such as theperfume, cosmetic and health care industries routinely use terpenesand/or terpenoid products as flavours and fragrances. By way of example,many sesquiterpene compounds are used in perfumery (e.g., patchoulol)and in the flavour industry (e.g., nootkatone) and many are extractedfrom plants. However, factors such as: (i) the availability and highprice of the plant raw material; (ii) the relatively low terpene contentin plant; and (iii) the tedious and inefficient extraction processes toproduce sufficient quantities of terpene products on an industrial scaleall have stimulated research on the biosynthesis of terpenes usingplant-independent systems. Consequently, effort has been expended indeveloping technologies to engineer microorganisms for convertingrenewable resources such as glucose into terpenoid products. Bycomparison with traditional methods, microorganisms have the advantageof fast growth without the need for land to sustain development.

Many microorganisms use either the methylerythritol 4-phosphate (MEP)pathway or the melavonate (MVA) pathway to supply intermediatesnecessary to produce terpenoid products. These MEP or MVA pathways caninclude an endogenous or an engineered MEP or MVA pathway or both. Adetailed understanding of isoprenoid pathway engineering andoptimization is disclosed in WO 2011/060057, US 2011/0189717, US2012/107893, U.S. Pat. No. 8,512,988 and Ajikumar et al (2010) Science330 70-74, which discloses the production of various terpenoid compoundsincluding sesquiterpene compounds such as nootkatone, which is anoxidised sesquiterpene produced from a valencene sesquiterpenesubstrate.

Nootkatone(4,4a,5,6,7,8-hexahydro-6-isopropenyl-4,4a-dimethyl-2(3II)-naphtalenone)is an important flavour constituent of grapefruit and is usedcommercially to flavour soft drinks and other beverages, as well asbeing used in perfumery. The conventional method for nootkatonepreparation is by oxidation of valencene (see, e.g., U.S. Pat. Nos.6,200,786 and 8,097,442). The starting material valencene is expensiveand thus methods that consume valencene are less commerciallyacceptable. Because of these drawbacks, there is a need for commerciallyfeasible and sustainable methods to prepare nootkatone and associatedproducts.

SUMMARY OF THE INVENTION

An object of the present disclosure is to provide sustainable productionof oxygenated sesquiterpene products. Specifically, the presentdisclosure provides enzyme catalysts for the ex vivo or in vivoproduction of certain oxygenated sesquiterpenes. In some embodiments,the disclosure provides host cells engineered for the biosynthesis ofthe oxygenated sesquiterpenes. Another object of the present disclosureis to provide engineered cytochrome P450 (CYP450) enzymes for synthesisof oxygenated sesquiterpenes, including in some embodiments functionalexpression alongside a reductase counterpart in E. coli, yeast, or otherhost cell. The disclosure thereby harnesses the unique capability ofthis class of enzymes to conduct oxidative chemistry.

In one aspect, the disclosure provides a method for making an oxygenatedproduct of a sesquiterpene. The method comprises contacting thesesquiterpene with Stevia rebaudiana Kaurene Oxidase (SrKO) or aderivative thereof having sesquiterpene oxidizing activity.Surprisingly, the wild type SrKO enzyme was shown to have activity on asesquiterpene substrate even though its natural activity is understoodto act on a diterpene substrate. Further, SrKO enzyme showed uniqueactivities, including oxygenation, by creating different stereoisomersof the hydroxylated product (alpha and beta nootkatol and furtheroxidizing to ketone, nootkatone), and produced different oxygenatedterpene products including hydroxygermacra-1(10)5-diene, andmurolan-3,9(11) diene-10-peroxy. This activity is distinct from otherP450 enzymes tested, which produced only one of the stereoisomers of thehydroxylated product (e.g., 0-nootkatol), as the major product andproduced only minor amounts of nootkatone. This activity of SrKOprovides a unique sesquiterpene oil for flavoring applications.

In some embodiments, the method takes place in an ex vivo (e.g., cellfree) system. In other embodiments, the sesquiterpene substrate and theSrKO or derivative thereof are contacted in a cell expressing the SrKO,such as a bacterium (e.g., E. coli). The oxygenated product of asesquiterpene may be recovered, or may be the substrate for furtherchemical transformation. Functional expression of wild type cytochromeP450 in E. coli has inherent limitations attributable to the bacterialplatforms (such as the absence of electron transfer machinery andcytochrome P450 reductases, and translational incompatibility of themembrane signal modules of P450 enzymes due to the lack of anendoplasmic reticulum). Thus, in some embodiments the SrKO enzyme ismodified for functional expression in an E. coli host cell, for example,by replacing a portion of the SrKO N-terminal transmembrane region witha short peptide sequence that stabilizes interactions with the E. coliinner membrane and/or reduces cell stress.

In some embodiments, the SrKO derivative has at least one mutation withrespect to the wild type SrKO that increases valencene oxidase activity,or increases production of nootkatone, α-nootkatol, and/or β-nootkatol.For example, the SrKO may have from 1 to 50 mutations independentlyselected from substitutions, deletions, or insertions relative to wildtype SrKO (SEQ ID NOS: 37 and 108) or an SrKO modified for expressionand activity in E. coli (e.g., SEQ ID NOS: 38 and 106). For example, theSrKO derivative may have from 1 to 40 mutations, from 1 to 30 mutations,from 1 to 20 mutations, or from 1 to 10 mutations relative to SrKO (SEQID NOS: 37 or 38). In these or other embodiments, the SrKO derivativemay comprise an amino acid sequence having at least 50% sequenceidentity, or at least 60% sequence identity, or at least 70% sequenceidentity, or at least 80% sequence identity, or at least 90% sequenceidentity to SrKO (SEQ ID NOS: 37 or 38), and has valencene oxidaseactivity. The SrKO in various embodiments maintains valencene oxidaseactivity, or has increased valencene oxidase activity as compared to thewild type enzyme in an ex vivo or bacterial system (e.g., E coli).Various mutations of SrKO which maintain or enhance valencene oxidaseactivity are listed in Tables 2.1, 2.2, 2.3 and 6. Thus, in variousembodiments, the SrKO may have at least 2, at least 3, at least 4, atleast 5, at least 6, at least 7, at least 8, at least 9, or at least 10mutations selected from Tables 2.1, 2.2, 2.3 and/or 6. Exemplaryderivatives of SrKO, also referred to herein as “valencene oxidase” or“VO” are represented by, for example, SEQ ID NOS: 104 and 105, which mayfurther be derivatized for improvements in desired activity. Mutationsmay be selected empirically for increases in oxygenated sesquiterpenetiter, or selected by in silico evaluation, or both.

In accordance with aspects of the disclosure, oxygenated sesquiterpeneproducts are obtainable by contacting a sesquiterpene substrate withStevia rebaudiana Kaurene Oxidase (SrKO) or derivative thereof havingvalencene oxidizing activity. Unlike other CYP450 enzymes, when a SrKOenzyme is used with valencene sesquiterpene substrate, it produces adifferent oxygenated terpene product profile that can includehydroxygermacra-1(10)5-diene, murolan-3,9(11) diene-10-peroxy,alpha-nootkatol, beta-nootkatol, and nootkatone. By comparison, otherCYP450's having the activity of hydroxylating valencene only producedone of the stereoisomers (beta nootkatol) and did not producesignificant amounts of the ketone (nootkatone), which requires twooxygenation cycles. See Table 4 and FIG. 7.

In various embodiments, the sesquiterpene substrate is (or thepredominant sesquiterpene substrate is) valencene, germacrene (A, B, C,D, or E), farnesene, farnesol, nootkatol, patchoulol, cadinene, cedrol,humulene, longifolene, and/or bergamotene, β-ylangene, β-santalol,β-santalene, α-santalene, α-santalol, β-vetivone, α-vetivone, khusimol,bisabolene, β-aryophyllene, longifolene; α-sinensal; α-bisabolol,(−)-β-copaene, (−)-α-copaene, 4(Z),7(Z)-ecadienal, cedrol, cedrene,cedrol, guaiol, (−)-6,9-guaiadiene, bulnesol, guaiol, ledene, ledol,lindestrene, and alpha-bergamotene. In some embodiments, the predominantsesquiterpene substrate is valencene, and the predominant oxygenatedproduct is nootkatone and/or nootkatol, which in some embodimentscomprises both α and β nootkatol.

The disclosure, when applied in vivo, is applicable to a wide array ofhost cells. In some embodiments, the host cell is a microbial host, suchas a bacterium selected from E. coli, Bacillus subtillus, or Pseudomonasputida; or a yeast, such as a species of Saccharomyces, Pichia, orYarrowia, including Saccharomyces cerevisiae, Pichia pastoris, andYarrowia lipolytica.

In some embodiments, the host cell produces isopentyl pyrophosphate(IPP), which acts as a substrate for the synthesis of the sesquiterpene.In some embodiments, the IPP is produced by metabolic flux through anendogenous or heterologous methylerythritol phosphate (MEP) or mevalonicacid (MVA) pathway. In some embodiments, the sesquiterpene is producedat least in part by metabolic flux through an MEP pathway, and whereinthe host cell has at least one additional copy of a dxs, ispD, ispF,and/or idi gene.

In some embodiments, the host cell expresses a farnesyl pyrophosphatesynthase (FPPS), which produces farnesyl pyrophosphate (FPP) from IPP orDMAPP. The host cell may further express a heterologous sesquiterpenesynthase to produce the desired sesquiterpene scaffold. For example, insome embodiments the cell expresses a valencene synthase. Severalvalencene synthase enzymes are known including Vitis vinifera valencenesynthase (VvVS) (SEQ ID NO: 1) or Citrus sinensis valencene synthase(CsVS) (SEQ ID NO: 12), which may be employed with the presentdisclosure, or alternatively a derivative of the VvVS or CsVS. Exemplaryderivative VvVS enzymes are disclosed herein. In certain embodiments,the sesquiterpene synthase is a valencene synthase selected from Vv1M1(SEQ ID NO: 3), Vv2M1 (SEQ ID NO: 5), Vv1M5 (SEQ ID NO: 7), Vv2M5 (SEQID NO: 9), VS2 (SEQ ID NO: 11), and VS3 (SEQ ID NO: 129), as disclosedherein.

The SrKO or derivative thereof acts on the sesquiterpene (e.g.,valencene) to produce the oxygenated terpene product. In someembodiments the SrKO is a fusion protein with a cytochrome P450reductase partner (e.g., SrCPR), allowing the cofactor to be efficientlyregenerated. In other embodiments, a P450 reductase is provided (e.g.,to in vitro system) or expressed in the host cell separately, and may beexpressed in the same operon as the SrKO in some embodiments. In someembodiments, the CPR enzyme is expressed separately, and the gene may beintegrated into the host cell genome in some embodiments. Variousexemplary CPR enzymes are disclosed herein, and which may be derivatizedto improve oxygenated sesquiterpenoid titer and/or to improve P450efficiency.

In some embodiments, the host cell expresses one or more enzymes thatfurther direct oxygenated product to nootkatone, such as the expressionof one or more alcohol dehydrogenase (ADH) enzymes. Exemplary ADHenzymes are disclosed herein.

In other aspects, the disclosure provides a method for making a productcontaining an oxygenated sesquiterpene, which comprises incorporatingthe oxygenated sesquiterpene prepared and recovered according to themethods described herein into a consumer or industrial product. Forexample, the product may be a flavor product, a fragrance product, acosmetic, a cleaning product, a detergent or soap, or a pest controlproduct. In some embodiments, the oxygenated product recovered comprisesnootkatol (e.g., α and/or β nootkatol) and/or nootkatone, and theproduct is a flavor product selected from a beverage, a chewing gum, acandy, or a flavor additive.

In other aspects, the disclosure provides engineered SrKO enzymes havingenhanced valencene oxidase activity as compared to wild type, as well ashost cells producing an oxygenated sesquiterpene as described herein,and which express all of the enzyme components for producing the desiredoxygenated sesquiterpene from isopentyl pyrophosphate (IPP). Forexample, the host cell in various embodiments expresses a farnesylpyrophosphate synthase, a sesquiterpene synthase, and the SrKO orderivative thereof. IPP may be produced through the MEP and/or MVApathway, which may be endogenous to the host cell, and which may beenhanced through expression of heterologous enzymes or duplication ofcertain enzymes in the pathway. Host cells include various bacteria andyeast as described herein. The oxygenated sesquiterpene (e.g.,nootkatone and/or nootkatol) may be recovered from the culture, and/oroptionally may act as the substrate for further chemical transformationin the cell or ex vivo system.

In another aspect, the disclosure provides sesquiterpene-containing oilproduced by the methods and host cells described herein. In someembodiments, the oil comprises hydroxygermacra-1(10)5-diene,murolan-3,9(11) diene-10-peroxy, alpha-nootkatol, beta-nootkatol, andnootkatone. In some embodiments, the predominant oxygenated products ofvalencene is nootkatone and nootkatol, and the oxygenated sesquiterpeneproduct comprises both alpha and beta nootkatol.

In another aspect, there is provided an SrKO crystal model structure(CMS) based on the structural coordinates of P45017A1 (which catalyzesthe biosynthesis of androgens). The CMS, including the terpene bindingpocket domain (TBD) that comprises a terpene binding pocket (TBP) and aterpene (e.g., valencene) bound to the TBD, is illustrated in FIGS. 8Aand 8B. This SrKO crystal model structure (CMS) facilitates in-silicotesting of SrKO derivatives. In part aided by this homology model, thepresent disclosure illustrates the use of several mutational strategiesto identify increases or improvements in sesquiterpene oxygenationactivity, including back-to-consensus mutagenesis, site-saturationmutagenesis, and recombination library screening.

Additional aspects and embodiments of the invention will be apparentfrom the following detailed description.

DESCRIPTION OF THE DRAWINGS

FIG. 1 shows a scheme for biosynthesis of valencene, which is asubstrate for SrKO in accordance with the present disclosure.

FIG. 2 depicts the fold productivities for site-directed mutants made toVvVS. 46 of the 225 point mutations convey an average improvement inproductivity of valencene of at least 20% compared to the wild-type WTVvVS. FIG. 2 shows the number of VvVS mutants (y-axis) exhibitingcertain levels of productivity (x-axis) versus the wild type.

FIG. 3 (A and B) provides the amino acid and nucleotide sequences ofvalencene synthases. FIG. 3A shows amino acid and nucleotide sequencesfrom Vitis vinfera wild-type (WT) (VvVS) (SEQ ID NOS: 1 and 2) andderivatives Vv1M1 (SEQ ID NOS: 3 and 4), Vv2M1 (SEQ ID NOS: 5 and 6),Vv1M5 (SEQ ID NOS: 7 and 8), Vv2M5 (SEQ ID NOS: 9 and 10), and VS2 (SEQID NOS: 11 and 120); as well as amino acid sequence for Citrus sinensiswild-type (CsVS) (SEQ ID NOS: 12 and 119). FIG. 3B shows an alignment ofwild-type VvVS and CsVS sequences, and the engineered Vv2M5 and VS2sequences.

FIG. 4 (A and B) provides the amino acid and nucleotide sequences ofvarious CYP450 (Cytochrome P450) enzymes having activity onsesquiterpene scaffolds. FIG. 4A shows sequences of wild type amino acidsequences and amino acid and nucleotide sequences engineered forbacterial expression: ZzHO (SEQ ID NO: 13, 14, and 15 respectively),BsGAO (SEQ ID NO: 16, 17, and 18, respectively), HmPO (SEQ ID NO: 19,20, and 21 respectively), LsGAO (SEQ ID NO: 22, 23, and 24,respectively), NtEAO (SEQ ID NO: 25, 26, and 27, respectively), CpVO(SEQ ID NO: 28, 29, and 30, respectively), AaAO (SEQ ID NO: 31, 32, and33, respectively), AtKO (SEQ ID NO: 34, 35, and 36 respectively), SrKO(SEQ ID NO: 37, 38, and 39 respectively), PpKO (SEQ ID NO: 40, 41, and42, respectively), BmVO (SEQ ID NO: 43 and SEQ ID NO: 44, respectively),PsVO (SEQ ID NO: 45 and SEQ ID NO: 46, respectively), PoLO (SEQ ID NO:47 and SEQ ID NO: 48, respectively), CiVO (SEQ ID NO: 49, 50, and 51respectively), HaGAO (SEQ ID NO: 52, 53, and 54, respectively). FIG. 4Bshows amino acid sequences of engineered Valencene Oxidase enzymes basedon the SrKO scaffold (SEQ ID NOS: 55-61).

FIGS. 5A and 5B depict construct designs for expression of MEP, terpeneand terpenoid synthases, and P450 enzymes in E. coli. FIG. 5A showsstrain configuration of upstream MEP pathway genes and the two plasmidsharboring downstream pathway genes. FIG. 5B shows construction of P450fusions, whereby N-terminal regions of both the P450 and CPR (CytochromeP450 reductase) are truncated and an exemplary leader sequence(MALLLAVF—SEQ ID NO:112) (8RP) is added while the two are fused with ashort linker peptide.

FIG. 6 (A-D) provides the amino acid and nucleotide sequences of variousCPR (Cytochrome P450 reductase) enzymes with sequence alignments. InFIG. 6A: Stevia rebaudiana (Sr)CPR (SEQ ID NOS: 62 and 63, Steviarebaudiana (Sr)CPR1 SEQ ID NOS: 76 and 77), Arabidopsis thaliana (At)CPR (SEQ ID NOS: 64 and 65), Taxus cuspidata (Tc) CPR (SEQ ID NOS: 66and 67), Artemisia annua (Aa)CPR) SEQ ID NOS: 68 and 69), Arabidopsisthaliana (At)CPR1 (SEQ ID NOS: 70 and 71), Arabidopsis thaliana (At)CPR2(SEQ ID NOS: 72 and 73), Arabidopsis thaliana (At)R2 (SEQ ID NOS: 74 and75); Stevia rebaudiana (Sr)CPR2 (SEQ ID NOS: 78 and 79); Steviarebaudiana (Sr)CPR3 (SEQ ID NOS: 80 and 81); Pelargonium graveolens(Pg)CPR (SEQ ID NO: 82 and 83). FIG. 6B shows an alignment of amino acidsequences for Arabidopsis thaliana and Artemisia annua CPR sequences(SEQ ID NOS:72, 74, 68, 64, and 70). FIG. 6C shows an alignment ofStevia rebaudiana CPR sequences (SEQ ID NOS: 78, 80, 62, and 76). FIG.6D shows an alignment of eight CPR amino acid sequences (SEQ ID NO: 74,72, 82, 68, 80, 62, 78, and 76).

FIG. 7 provides GC-chromatographs which show the different activities ofvarious CYP450 enzymes, as expressed in valencene-producing E. colialong with CPR partners as described in Example 2. Strains were culturedfor four days and extracted with Methyl Tert-Butyl Ether (MTBE). 1 μl ofMTBE was injected through GC-MS and the product profiles were monitoredby comparing with a MS library. From top to bottom: Taxus 5-alphahydroxylase, Cichorium intybus (CiVO) P450 (SEQ ID NO:50), Hyoscyamusmuticus (HmPO) P450 (SEQ ID NO:20), and SrKO (SEQ ID NO:38).

FIGS. 8A and 8B illustrate a homology model of SrKO and its active site.The SrKO homology model is based on the known mutant P45017A1 (thecrystal structure of membrane-bound cytochrome P450 17 A1 as disclosedin DeVore N M and Scott E E (Nature, 482, 116-119, 2012), whichcatalyzes the biosynthesis of androgens in human. The position of theheme is shown as sticks. FIG. 8B depicts a structural model of SrKOactive site with valencene docked in its α-binding mode. Secondarystructure motifs (B-C Loop and I-Helix) and amino acids targeted formutagenesis are shown.

FIG. 9 shows optimizing the valencene oxidase (VO) N-terminal membraneanchor. The N-terminus of E. coli yhcB was selected as a membrane anchorsequence, which provides a single-pass transmembrane helix. The lengthof the anchor (from 20 to 24 amino acids) and the VO N-terminaltruncation length (from 28 to 32 amino acids) were screened forimprovements in oxygenation titer.

FIG. 10 shows that a truncation length of 29, and a 20 amino acidN-terminal anchor based on E. coli yhcB, led to a 1.2-fold increase intotal oxygenated titer compared to the average of controls.

FIG. 11 illustrates an exemplary downstream pathway for expression inthe host cell, for conversion of farnesyl diphosphate to nootkatone.Farnesyl diphosphate (produced from IPP/DMAPP by an expressed FarnesylPyrophosphate Synthase) is converted to valencene by the action of aValencene Synthase (VS), which is oxidized by a Valencene Oxidase (VO),such as SrKO or an SrKO derivative described herein. The VO cofactor isregenerated by a cytochrome P450 reductase (CPR). The products ofoxidation by VO can include nootkatol (α and β) and nookatone, which canbe further directed to nootkatone by the action of an AlcoholDehydrogenase (ADH).

FIG. 12 (A and B) shows the oxygenation profile for a strain expressingVO1-L-SrCPR. The oxygenation profile includes the single oxygenationproducts of β-nootkatol and α-nootkatol along with the two-stepoxygenation product, nootkatone. Panel (A) shows the profile in mg/L.Panel (B) shows the profile by percent of total oxygenated product (thelegend for panels (A) and (B) are the same).

FIG. 13 (A and B) shows evaluation of mutations identified using aback-to-consensus strategy in wild-type SrKO, translated into anengineered valencene oxidase background (n22yhcB_t30VO1 (SEQ ID NO:104)). More than 50% of the mutations resulted in a 1.2 to 1.45 foldimprovement in total oxygenated titers. Panel (A) shows titer in mg/L.Panel (B) shows fold change in oxygenated titer and ratio of a/Pnootkatol.

FIG. 14 shows results of secondary screening of back-to-consensusmutations, N-terminal anchor optimization, and site-saturationmutagenesis (SSM). Several mutations were identified that show a 1.1 to1.4-fold improvement in oxygenated titers.

FIG. 15 shows performance of select VO1 variants at 33° C. Six mutationswere identified that maintained improved productivities at 33° C.

FIG. 16 (A and B) shows results from primary screening of therecombination library. Several variants (shown) exhibited up to1.35-fold improvement in oxygenated product titer. There was a shift inprofile to more (+)-nootkatone and higher oxygenation capacity forselect variants. Panel (A) shows oxygenated product in mg/L. Panel (B)plots the fold change in oxygenation capacity (nootkatols require onlyone oxygenation cycle from valencene, while nootkatone requires twooxygenation cycles).

FIG. 17 shows oxygenation capacity at 34° C. and 37° C. for select VOrecombination library variants.

FIG. 18 shows oxygenation titer at 34° C. and 37° C. after re-screen oflead VO variants. C6(1) (R76K, M94V, T131Q, I390L, T468I) had thehighest oxygenation capacity at 37° C., and was designated VO2 (SEQ IDNO: 111).

FIG. 19 shows screening of cytochrome P450 reductase (CPR) orthologs forenhanced valencene oxidase activity (30° C.). SrCPR3 shows increasedoxygenation titer and higher production of Nootkatone.

FIG. 20 shows screening of CPR orthologs at 34° C. SrCPR3 and AaCPRexhibit ˜1.3-fold improvement in oxygenated titer, even at the highertemperature.

FIG. 21 shows conversion of nootkatols to nootkatone with an alcoholdehydrogenase (ADH). Four ADH orthologs (vvDH, csABA2, bdDH, and zzSDR)were identified that convert β-nootkatol to (+)-nootkatone, resulting ina 3-fold increase in (+)-nootkatone titers and an increase in proportionof α-nootkatol.

FIG. 22 (A and B) depicts alcohol dehydrogenase enzymes. FIG. 22A showsamino acid and nucleotide sequences including those for Rhodococcuserythropolis (Re)CDH (SEQ ID NOS: 84 and 85), Citrus sinensis (Cs)DH(SEQ ID NOS: 86 and 87), Citrus sinensis (Cs)DH1 (SEQ ID NOS: 88 and89), Citrus sinensis (Cs)DH2 (SEQ ID NOS: 90 and 91), Citrus sinensis(Cs)DH3 (SEQ ID NOS: 92 and 93), Vitis vinifera (Vv)DH (SEQ ID NOS: 94and 95), Vitis vinifera (Vv)DH1 (SEQ ID NOS: 96 and 97, Citrus sinensis(Cs)ABA2 (SEQ ID NOS: 98 and 99), Brachypodium distachyon (Bd)DH (SEQ IDNO: 100 and 101), Zingiber zerumbet (Zz)SDR (SEQ ID NOS: 102 and 103).FIG. 22B shows an alignment of the amino acid sequences.

FIG. 23 (A and B) shows alignments of several engineered valenceneoxidase (VO) variants. In FIG. 23A: 8rp-t20SrKO (SEQ ID NO: 106) is theSrKO sequence with a 20-amino acid truncation at the N-terminus, and theaddition of an 8-amino acid membrane anchor. 8rp-t20VO0 (SEQ ID NO: 107)has a truncation of 20 amino acids of the SrKO N-terminus, the additionof an 8-amino acid N-terminal anchor, and a single mutation at position499 (numbered according to wild-type SrKO). n22yhcB-t30VO1 (SEQ ID NO:104) has a 30-amino acid truncation of the SrKO N-terminus, a membraneanchor based on 22 amino acids from E. coli yhcB, and eight pointmutations at positions 46, 231, 284, 383, 400, 444, 488, and 499 (withrespect to SrKO wild-type). n22yhcB-t30VO2 (SEQ ID NO: 105) has a30-amino acid truncation of the SrKO N-terminus, a membrane anchor basedon 22 amino acids from E. coli yhcB, and nine point mutations atpositions 76, 94, 131, 231, 284, 383, 390, 468, and 499 (with respect toSrKO wild-type). In FIG. 23B, point mutations in VO0 (SEQ ID NO: 109),VO1 (SEQ ID NO: 110), and VO2 (SEQ ID NO: 111) are shown againstwild-type SrKO (SEQ ID NO: 108) (all shown with the wild-type SrKON-terminus for convenience).

DETAILED DESCRIPTION OF THE INVENTION

The present disclosure in various aspects provides methods for makingoxygenated terpenes or terpenoids in ex vivo or in cell systems. Thedisclosure further provides engineered or modified enzymes,polynucleotides, and host cells for use in such methods. The disclosurein various embodiments is directed to a method to produce nootkatoneand/or nootkatol using an SrKO enzyme. Surprisingly, it was found thatthe SrKO enzyme can be used to catalyze sesquiterpene oxidation (e.g.,valencene oxidation to nootkatol and nootkatone).

As used herein, SrKO refers to ent-kaurene oxidase CYP701A5 [Steviarebaudiana] with Accession No AAQ63464.1 (SEQ ID NO: 37). SrKO and itsactivity on diterpenes (and kaurene in particular) are known and aredescribed in, for example, US 2012/0164678, which is hereby incorporatedby reference in its entirety. It is a member of the CYP70 family ofcytochrome p450 enzymes (CYP450). An exemplary SrKO sequence modifiedfor expression in E. coli is shown as SEQ ID NO: 38. As shown herein,SrKO is active on sesquiterpene substrates (e.g., valencene), producingnootkatol (both α and β) and nootkatone, which are valuable terpenoidcompounds. Further, SrKO provides a unique product profile with uniquesensory characteristics that is based on the oxygenation of valencene.These activities and product profiles can be further refined bymutagenesis of the SrKO using processes (and aided by in silico models)described in detail herein.

As used herein, the term “SrKO derivative,” “modified SrKO polypeptide,”“engineered SrKO,” “SrKO variant,” “engineered valencene oxidase,” or“valencene oxidase variant” refers to an amino acid sequence that hassubstantial structural and/or sequence identity with SrKO, and catalyzesoxygenation of a sesquiterpene scaffold, such as valencene. SrKO enzymesengineered for the oxygentation of valencene are also referred to hereinas “valencene oxidase” or “VO” enzymes. Generally, derivatives comprisemutated forms of SrKO having at least one mutation that increases theactivity of the enzyme for the valencene substrate or for the productionof nootkatone, nootkatol, and/or other products. Some SrKO mutations areprovided in Tables 2.1, 2.2, and 2.3. Some such additional SrKOmutations are provided in Table 6.

The term “contacting” means that the components are physically broughttogether, whether in vivo through co-expression of relevant proteinproducts (e.g., sesquiterpene synthase and CYP450) in a host cell, or byadding or feeding a substrate of interest to a host cell expressing anSrKO or derivative thereof, or in vitro (or “ex vivo”) by addingsesquiterpene substrate to purified P450 enzyme or cellular extract orpartially purified extract containing the same. The terms in vitro andex vivo refer to a cell free system, and may be performed in a reactiontube or well.

As used herein, “terpenes” are a large and varied class of hydrocarbonsthat have a simple unifying feature, despite their structural diversity.According to the “isoprene rule”, all terpenes consist of isoprene (C5)units. This fact is used for a rational classification depending on thenumber of such units. Monoterpenes comprise 2 isoprene units and areclassified as (C10) terpenes, sesquiterpenes comprise 3 isoprene unitsand are classified as (C15) terpenes, diterpenes comprise 4 isopreneunits and are classified as (C20) terpenes, sesterterpenes (C25),triterpenes (C30) and rubber (C5)n. They occur as acyclic or mono- topentacyclic derivatives with alcohol, ether, ester, aldehyde, or ketonegroups (the so called “terpenoids”), everywhere in organisms,particularly in higher plants, and are characteristic of the individualtype of plants. Terpenes such as Monoterpenes (C10), Sesquiterpenes(C15) and Diterpenes (C20) are derived from the prenyl diphosphatesubstrates, geranyl diphosphate (GPP), farnesyl diphosphate (FPP) andgeranylgeranyl diphosphate (GGPP) respectively through the action of avery large group of enzymes called the terpene (terpenoid) synthases.These enzymes are often referred to as terpene cyclases since theproduct of the reactions are cyclised to various monoterpene,sesquiterpene and diterpene carbon skeleton products. Many of theresulting carbon skeletons undergo subsequence oxygenation by cytochromep450 hydrolysase enzymes to give rise to large families of derivatives.The technical syntheses of top-selling flavours and fragrances can startfrom terpenes which can also serve as excellent solvents or dilutingagents for dyes and varnishes. Natural or synthetic resins of terpenesare used and also many pharmaceutical syntheses of vitamins andinsecticides start from terpenes. As used herein, the term “terpene” or“sesquiterpene” (for example) includes corresponding terpenoid orsesquiterpenoid compounds.

As used herein, the term “oxygenated sesquiterpene” refers to asesquiterpene scaffold having one or more oxygenation events, producinga corresponding alcohol, aldehyde, carboxylic acid and/or ketone. One ormore oxygenated sesquiterpenes may be referred to herein as an“oxygenated product.”

As used herein, the term “unoxygenated sesquiterpene” refers to asesquiterpene scaffold that has not undergone any oxygenation events. Anunoxygenated sesquiterpene may also be referred to herein as an“unoxygenated product.” As used herein, the term “oxygenated producttiter” or “oxygenated titer” refers to the sum of titers of α-nootkatol,β-nootkatol, and (+)-nootkatone.

As used herein, the term “MEP pathway” refers to the(2-C-methyl-D-erythritol 4-phosphate) pathway, also called the MEP/DOXP(2-C-methyl-D-erythritol 4-phosphate/1-deoxy-D-xylulose 5-phosphate)pathway or the non-mevalonate pathway or the mevalonic acid-independentpathway. In the MEP pathway, pyruvate and D-glyceraldehyde-3-phosphateare converted via a series of reactions to IPP and DMAPP. The pathwaytypically involves action of the following enzymes:1-deoxy-D-xylulose-5-phosphate synthase (Dxs),1-deoxy-D-xylulose-5-phosphate reductoisomerase (IspC),4-diphosphocytidyl-2-C-methyl-D-erythritol synthase (IspD),4-diphosphocytidyl-2-C-methyl-D-erythritol kinase (IspE),2C-methyl-D-erythritol 2,4-cyclodiphosphate synthase (IspF),1-hydroxy-2-methyl-2-(E)-butenyl 4-diphosphate synthase (IspG), andisopentenyl diphosphate isomerase (IspH). The MEP pathway, and the genesand enzymes that make up the MEP pathway, are described in U.S. Pat. No.8,512,988, which is hereby incorporated by reference in its entirety.For example, genes that make up the MEP pathway include dxs, ispC, ispD,ispE, ispF, ispG, ispH, idi, and ispA.

As used herein, the “MVA pathway” refers to the biosynthetic pathwaythat converts acetyl-CoA to IPP. The mevalonate pathway typicallycomprises enzymes that catalyze the following steps: (a) condensing twomolecules of acetyl-CoA to acetoacetyl-CoA (e.g., by action ofacetoacetyl-CoA thiolase); (b) condensing acetoacetyl-CoA withacetyl-CoA to form hydroxymethylglutaryl-CoenzymeA (HMG-CoA) (e.g., byaction of HMG-CoA synthase (HMGS)); (c) converting HMG-CoA to mevalonate(e.g., by action of HMG-CoA reductase (HMGR)); (d) phosphorylatingmevalonate to mevalonate 5-phosphate (e.g., by action of mevalonatekinase (MK)); (e) converting mevalonate 5-phosphate to mevalonate5-pyrophosphate (e.g., by action of phosphomevalonate kinase (PMK)); and(f) converting mevalonate 5-pyrophosphate to isopentenyl pyrophosphate(e.g., by action of mevalonate pyrophosphate decarboxylase (MPD)). TheMVA pathway, and the genes and enzymes that make up the MEP pathway, aredescribed in U.S. Pat. No. 7,667,017, which is hereby incorporated byreference in its entirety.

As used herein, the term “cytochrome P450 reductase partner” or “CPRpartner” refers to a cytochrome P450 reductase capable of regeneratingthe cofactor component of the cytochrome P450 oxidase of interest (e.g.,SrKO) for oxidative chemistry. For example, SrCPR is a natural CPRpartner for SrKO. In some embodiments, the CPR partner is not thenatural CPR partner for SrKO. In some embodiments employing in vivoproduction of oxygenated sesquiterpene, the SrKO and SrCPR areco-expressed as separate proteins, or in some embodiments are expressedas a fusion protein.

As used herein, the term “natural product” refers to a product obtained,at least in part, from plant and/or animal material or obtained frommicrobial enzymatic biotransformations/bioconversions/biocatalysisand/or biosynthesis.

Ranges can be expressed herein as from “about” one particular value,and/or to “about” another particular value. When such a range isexpressed, another embodiment includes from the one particular valueand/or to the other particular value. Similarly, when values areexpressed as approximations, by use of the antecedent “about,” it willbe understood that the particular value forms another embodiment. Itwill be further understood that the endpoints of each of the ranges aresignificant both in relation to the other endpoint, and independently ofthe other endpoint. It is also understood that there are a number ofvalues disclosed herein, and that each value is also herein disclosed as“about” that particular value in addition to the value itself. Forexample, if the value “10” is disclosed, then “about 10” is alsodisclosed. It is also understood that each unit between two particularunits are also disclosed. For example, if 10 and 15 are disclosed, then11, 12, 13, and 14 are also disclosed.

The similarity of nucleotide and amino acid sequences, i.e., thepercentage of sequence identity, can be determined via sequencealignments. Such alignments can be carried out with several art-knownalgorithms, such as with the mathematical algorithm of Karlin andAltschul (Karlin & Altschul (1993) Proc. Natl. Acad. Sci. USA 90:5873-5877), with hmmalign (HMMER package, hmmer.wustl.edu/) or with theCLUSTAL algorithm (Thompson, J. D., Higgins, D. G. & Gibson, T. J.(1994) Nucleic Acids Res. 22, 4673-80). The grade of sequence identity(sequence matching) may be calculated using e.g. BLAST, BLAT or BlastZ(or BlastX). A similar algorithm is incorporated into the BLASTN andBLASTP programs of Altschul et al (1990) J. Mol. Biol. 215: 403-410.BLAST polynucleotide searches can be performed with the BLASTN program,score=100, word length=12.

BLAST protein searches may be performed with the BLASTP program,score=50, word length=3. To obtain gapped alignments for comparativepurposes, Gapped BLAST is utilized as described in Altschul et al (1997)Nucleic Acids Res. 25: 3389-3402. When utilizing BLAST and Gapped BLASTprograms, the default parameters of the respective programs are used.Sequence matching analysis may be supplemented by established homologymapping techniques like Shuffle-LAGAN (Brudno M., Bioinformatics 2003b,19 Suppl 1:154-162) or Markov random fields.

“Conservative substitutions” may be made, for instance, on the basis ofsimilarity in polarity, charge, size, solubility, hydrophobicity,hydrophilicity, and/or the amphipathic nature of the amino acid residuesinvolved. The 20 naturally occurring amino acids can be grouped into thefollowing six standard amino acid groups:

(1) hydrophobic: Met, Ala, Val, Leu, He;

(2) neutral hydrophilic: Cys, Ser, Thr; Asn, Gin;

(3) acidic: Asp, Glu;

(4) basic: His, Lys, Arg;

(5) residues that influence chain orientation: Gly, Pro; and

(6) aromatic: Trp, Tyr, Phe.

As used herein, “conservative substitutions” are defined as exchanges ofan amino acid by another amino acid listed within the same group of thesix standard amino acid groups shown above. For example, the exchange ofAsp by Glu retains one negative charge in the so modified polypeptide.In addition, glycine and proline may be substituted for one anotherbased on their ability to disrupt a-helices. Some preferred conservativesubstitutions within the above six groups are exchanges within thefollowing sub-groups: (i) Ala, Val, Leu and He; (ii) Ser and Thr; (ii)Asn and Gin; (iv) Lys and Arg; and (v) Tyr and Phe.

As used herein, “non-conservative substitutions” or “non-conservativeamino acid exchanges” are defined as exchanges of an amino acid byanother amino acid listed in a different group of the six standard aminoacid groups (1) to (6) shown above.

In one aspect, the disclosure provides a method for making an oxygenatedproduct of a sesquiterpene. In various embodiments, the sesquiterpenesubstrate is (or the predominant sesquiterpene substrate is) valencene,germacrene (A, B, C, D, or E), farnesene, farnesol, nootkatol,patchoulol, cadinene, cedrol, humulene, longifolene, and/or bergamotene,β-ylangene, β-santalol, β-santalene, α-santalene, α-santalol,β-vetivone, α-vetivone, khusimol, bisabolene, β-aryophyllene,Longifolene; α-sinensal; α-bisabolol, (−)-β-copaene, (−)-α-copaene,4(Z),7(Z)-ecadienal, cedrol, cedrene, cedrol, guaiol,(−)-6,9-guaiadiene, bulnesol, guaiol, ledene, ledol, lindestrene, andalpha-bergamotene. In some embodiments, the predominant sesquiterpenesubstrate is valencene, and the predominant oxygenated product isnootkatone and/or nootkatol, which in some embodiments comprises both αand β nootkatol. In this context, the term “predominant” means that theparticular sesquiterpene is present at a level higher than all otherterpene or terpenoid species individually. In some embodiments, thepredominant sesquiterpene (either the substrate or the oxygenatedproduct after the reaction) makes up at least 25%, at least 40%, atleast 50%, or at least 75% of the terpene or terpenoid component of thecomposition. In various embodiments involving in vivo production ofoxygenated sesquiterpenes, the oxygenated product is recovered from theculture media, and can be fractionated to isolate or enrich for variouscomponents of the product, such as nootkatone, nootkatol, and/or othercomponents.

In various embodiments, the disclosure comprises contacting asesquiterpene with a terpene oxidizing P450 enzyme, or a derivativethereof. The contacting may take place in a host cell or in a cell freesystem. The substrate for oxidation (e.g., the sesquiterpene), may beproduced by the cells (e.g., through metabolic flux through the MEP orMVA pathways), or alternatively fed to the host cells expressing theP450 enzyme. The oxygenated product may be recovered, or be thesubstrate for further chemical transformation either in the cellularsystem or cell free system. Table 1 below provides a list of exemplaryP450 enzymes. While in certain embodiments the disclosure involves theuse of the following P450 enzymes (optionally engineered to increase theoxygenation of valencene to nootkatone and/or nootkatol), a preferredenzyme in accordance with this disclosure is SrKO. Exemplary oxygenatedsesquiterpene products obtained by these reactions in accordance withthe disclosure are shown in Table 4.

TABLE 1 # Species Name Native Substrate Native Reaction Product 1Zingiber zzHO α-humulene 8-hydroxy-α-humulene zerumbet 2 BarnadesiaBsGAO germacrene A germacra-1(10),4,11(13)- spinosa trien-12-ol 3Hyoscyamus HmPO premnaspirodiene solavetivol muticus 4 Latuca LsGAOgermacrene A germacra-1(10),4,11(13)- spicata trien-12-ol 5 NicotianaNtEAO 5-epi-aristolochene capsidiol tabacum 6 Citrus x CpVO valencenenootkatol paradisi 7 Artemesia AaAO amorphadiene artemisinic acid annua8 Arabidopsis AtKO kaurene kaurenoic acid thaliana 9 Stevia SrKO kaurenekaurenoic acid rebaudiana 10 Pseudomonas PpKO kaurene kaurenoic acidputida 11 Bacillus BmVO fatty acids hydroxylated FAs megateriurn 12Pleurotus PsVO valencene nootkatone sapidus 13 Pleurotus PoLO unknownunknown ostreatus 14 Cichorium CiVO valencene nootkatone intybus 15Helianthus HaGAO germacrene A germacrene A acid annuus

In various embodiments, the method comprises contacting thesesquiterpene with a protein comprising Stevia rebaudiana KaureneOxidase (SrKO) or derivative thereof. In some embodiments the SrKO isexpressed in a host cell as described below, or is provided in a cellfree system. For example, certain in vitro and in vivo systems foroxidizing terpenes with P450 enzymes are disclosed in U.S. Pat. No.7,211,420, which are hereby incorporated by reference. McDougle D R,Palaria A, Magnetta E, Meling D D, Das A. Functional Studies ofN-terminally modified CYP2J2 epoxygenase in Model Lipid Bilayers,Protein Sci. 2013 22:964-79; Luthra, A., Gregory, M., Grinkova, Y. V.,Denisov, I. G., Sligar, S. G. (2013) “Nanodiscs in the studies ofmembrane-bound cytochrome P450 enzymes.” Methods Mol. Biol., 987,115-127).

In some embodiments, the SrKO derivative comprises an amino acidsequence that has from about 1 to about 50 mutations independentlyselected from substitutions, deletions, or insertions relative to SrKO(SEQ ID NO: 37 or 38), or relative to an SrKO enzyme modified at itsN-terminus for functional expression in E. coli (SEQ ID NO: 38 or 55).In various embodiments, the mutation or combination of mutationsenhances the activity of the enzyme for oxygenation of valencene, suchas the production of nootkatone and/or nootkatol. Protein modeling asdescribed herein may be used to guide such substitutions, deletions, orinsertions in the SrKO sequence. For example, a structural model of theSrKO amino acid sequence may be created using the coordinates forP45017A1. As demonstrated herein, such a homology model is useful fordirecting improvement of SrKO for valencene oxygenation. Thus, invarious embodiments, the SrKO derivative may have from about 1 to about45 mutations, about 1 to about 40 mutations, about 1 to about 35mutations, from about 1 to about 30 mutations, about 1 to about 25mutations, from about 1 to about 20 mutations, about 1 to about 15mutations, about 1 to about 10 mutations, or from about 1 to about 5mutations relative to SrKO (SEQ ID NO: 37, 38, or 55). In variousembodiments, the SrKO comprises a sequence having at least 5 or at least10 mutations with respect to SEQ ID NO: 37, 38, or 55, but not more thanabout 20 or 30 mutations. In various embodiments, the SrKO derivativemay have about 1 mutation, about 2 mutations, about 3 mutations, about 4mutations, about 5 mutations, about 6 mutations, about 7 mutations,about 8 mutations, about 9 mutations, about 10 mutations, about 11mutations, about 12 mutations, about 13 mutations, about 14 mutations,about 15 mutations, about 16 mutations, about 17 mutations, about 18mutations, about 19 mutations, about 20 mutations, about 21 mutations,about 22 mutations, about 23 mutations, about 24 mutations, about 25mutations, about 26 mutations, about 27 mutations, about 28 mutations,about 29 mutations, about 30 mutations, about 31 mutations, about 32mutations, about 33 mutations, about 34 mutations, about 35 mutations,about 36 mutations, about 37 mutations, about 38 mutations, about 39mutations, about 40 mutations, about 41 mutations, about 42 mutations,about 43 mutations, about 44 mutations, about 45 mutations, about 46mutations, about 47 mutations, about 48 mutations, about 49 mutations,or about 50 mutations relative to SrKO (SEQ ID NO: 37, 38, or 55). SEQID NO: 37, and other WT enzymes disclosed herein, can optionally containan Ala inserted at position 2 where not present in the wild-type.

In these or other embodiments, the SrKO derivative may comprise an aminoacid sequence having at least about 50% sequence identity, at leastabout 55% sequence identity, at least about 60% sequence identity, atleast about 65% sequence identity, at least about 70% sequence identity,at least about 75% sequence identity, at least about 80% sequenceidentity, at least about 85% sequence identity, or at least 90% sequenceidentity, or at least 91% sequence identity, or at least 92% sequenceidentity, or at least 93% sequence identity, or at least 94% sequenceidentity, or at least 95% sequence identity, or at least 96% sequenceidentity, or at least 97% sequence identity, or at least 98% sequenceidentity, or at least 99% sequence identity, to SrKO (SEQ ID NO: 37, 38,or 55). In various embodiments, the SrKO derivative has higher activityfor the oxygenation of valencene than the wild type enzyme, such as ahigher production of oxygenated oil upon contact with valencenesubstrate than the wild type enzyme (SEQ ID NO: 37) or the wild typeenzyme as modified for functional expression in E. coli (e.g., SEQ IDNO: 38). For example, the SrKO derivative may comprise an amino acidsequence having at least: about 50% identity, about 51% identity, about52% identity, about 53% identity, about 54% identity, about 55%identity, about 56% identity, about 57% identity, about 58% identity,about 59% identity, about 60% identity, about 61% identity, about 62%identity, about 63% identity, about 64% identity, about 65% identity,about 66% identity, about 67% identity, about 68% identity, about 69%identity, about 70% identity, about 71% identity, about 72% identity,about 73% identity, about 74% identity, about 75% identity, about 76%identity, about 77% identity, about 78% identity, about 79% identity,about 80% identity, about 81% identity, about 82% identity, about 83%identity, about 84% identity, about 85% identity, about 86% identity,about 87% identity, about 88% identity, about 89% identity, about 90%identity, about 91% sequence identity, about 92% sequence identity,about 93% sequence identity, about 94% sequence identity, about 95%sequence identity, about 96% sequence identity, about 97% sequenceidentity, about 98% sequence identity, or about 99% sequence identity toSrKO (SEQ ID NO: 37, 38, or 55).

In some embodiments, mutants are selected for an increase in productionof oxygenated valencene, such as nootkatone, α-nootkatol, and/orβ-nootkatol. For example, the SrKO derivative may have one or moremutations at positions selected from 46, 76, 94, 131, 231, 284, 383,390, 400, 444, 468, 488 and 499, numbered according to SEQ ID NO: 37.For example, in some embodiments, the SrKO is a derivative comprising anamino acid sequence having one or more (or all) of the mutationsselected from H46R, R76K, M94V, T131Q, F231L, H284Q, R383K, I390L,V400Q, I444A, T468I, T488D, and T499N, numbered according to SEQ ID NO:37. In certain embodiments, the SrKO is a derivative comprising an aminoacid sequence having one or more (or all) of the mutations selected fromR76K, M94V, T131Q, F231L, H284Q, R383K, I390L, T468I, and T499N,numbered according to SEQ ID NO: 37. In some embodiments, the SrKOderivative comprises an amino acid sequence selected from SEQ ID NOS:55-61, which were engineered according to this disclosure to improveactivity for oxygenation of valencene (e.g., production of nootkatone).In some embodiments, the derivative comprises an amino acid sequencehaving from one to twenty mutations relative to a sequence selected fromSEQ ID NOS: 55-61, with the proviso that the amino acid sequence has oneor more mutations at positions selected from 46, 76, 94, 131, 231, 284,383, 390, 400, 444, 468, 488, and 499 (numbered according to SEQ ID NO:37), or the proviso that the SrKO derivative comprises an amino acidsequence having one or more (or all) of the mutations selected fromH46R, R76K, M94V, T131Q, F231L, H284Q, R383K, I390L, V400Q, I444A,T468I, T488D, and T499N (numbered according to SEQ ID NO: 37). Incertain embodiments, the derivative comprises an amino acid sequencehaving from one to twenty mutations relative to a sequence selected fromSEQ ID NOS: 55-61, with the proviso that the amino acid sequence has oneor more mutations at positions selected from 46, 76, 94, 131, 231, 284,383, 390, 400, 444, 468, 488, and 499 (numbered according to SEQ ID NO:37), or the proviso that the SrKO derivative comprises an amino acidsequence having one or more (or all) of the mutations selected fromR76K, M94V, T131Q, F231L, H284Q, R383K, I390L, T468I, and T499N(numbered according to SEQ ID NO: 37).

In some embodiments, the disclosure provides a recombinantpolynucleotide encoding the SrKO derivative described above, which maybe inserted into expression vectors for expression and optionalpurification. In some embodiments, the polynucleotide is incorporatedinto the genome of valencene-producing cells, such asvalencene-producing E. coli cells.

The SrKO or derivative in various embodiments has valencene oxidaseactivity. Assays for determining and quantifying valencene oxidaseactivity are described herein and are known in the art. Assays includeexpressing the SrKO (or derivative) in valencene-producing cells (e.g.,E. coli expressing FPPS and valencene synthase), and extracting theoxygenated oil from the aqueous reaction media. The profile of terpenoidproduct can be determined quantitatively by GC/MS. Various mutations ofSrKO tested for effect on valencene oxidase activity are listed inTables 2.1, 2.2, 2.3 and/or 6. Thus, in various embodiments, the SrKOmay have at least about 1, at least about 2, at least about 3, at leastabout 4, at least about 5, at least about 6, at least about 7, at leastabout 8, at least about 9, or at least about 10 mutations selected fromTables 2.1, 2.2, 2.3 and/or 6. In some embodiments, the SrKO derivativeis a modified SrKO polypeptide comprising an amino acid sequence whichhas up to 25 mutations compared to the wild type protein according toSEQ ID NO: 37 (or its counterpart that is modified for expression in E.coli), and comprises at least the substitutions 1310V, V375I or T487N incombination with at least any one or more of V375F, V375A, V375M, M120L,M120I, M120V, F129L, F129I, L114V, L114F and V121A (numbered accordingto SEQ ID NO: 38) (see Table 6), and optionally comprises a leadersequence (as shown in SEQ ID NO: 38) supporting functional expression inE. coli.

TABLE 2.1 Summary of some Stevia rebaudiana kaurene oxidase mutationstested, numbered according to wild type SrK0 (SEQ ID NOS: 37 and 108),8rp-t20SrKO (SEQ ID NOS: 38 and 106), n22yhcB-t30VO1 (SEQ ID NO: 104),and n22yhcB-t30VO2 (SEQ ID NOS: 61 and 105). Mutation (relative to SEQPosition Position ID NO: 37 (SEQ SEQ ID NO: 37 (SEQ SEQ ID NO: 104 / IDNO: 108) / ID NO: 108) / SEQ ID NO: SEQ ID NO: 38 SEQ ID NO: 38 (SEQ 105(SEQ ID NO: (SEQ ID NO: No. WT ID NO: 106) 61) 106) 1 L 59 / 47 51 I 2 Y71 / 59 63 H 3 M 72 / 60 64 K 4 T 75 / 63 67 A 5 A 79 / 67 71 E 6 K 88 /76 80 R 7 T 92 / 80 84 C 8 M 94 / 82 86 V 9 V 97 / 85 89 L 10 V 97 / 8589 I 11 S 98 / 86 90 N 12 Q 112 / 100 104 S 13 N 118 / 106 110 K 14 K124 / 112 116 T 15 A 128 / 116 120 R 16 T 131 / 119 123 S 17 M 135 / 123127 T 18 M 135 / 123 127 Q 19 M 135 / 123 127 F 20 M 135 / 123 127 T 21D 139 / 127 131 G 22 Y 141 / 129 133 F 23 A 152 / 140 144 R 24 K 161 /149 153 R 25 H 162 / 150 154 F 26 N 183 / 171 175 D 27 L 192 / 180 184 F28 I 195 / 183 187 V 29 D 220 / 208 212 E 30 D 244 / 232 236 E 31 S 279/ 267 271 A 32 H 284 / 272 276 Q 33 S 296 / 284 288 C 34 I 298 / 286 290L 35 Q 306 / 294 298 K 36 Q 311 / 299 303 E 37 I 322 / 310 314 T 38 I322 / 310 314 V 39 R 383 / 371 375 K 40 R 383 / 371 375 I 41 V 387 / 375379 T 42 V 387 / 375 379 I 43 V 387 / 375 379 L 44 I 390 / 378 382 V 45H 394 / 382 386 Y 46 V 400 / 388 392 Q 47 V 400 / 388 392 M 48 H 405 /393 397 D 49 L 412 / 400 404 I 50 V 425 / 413 417 D 51 V 425 / 413 417 K52 F 446 / 434 438 L 53 G 454 / 442 446 A 54 S 462 / 450 454 A 55 L 466/ 454 458 M 56 G 472 / 460 464 A 57 M 476 / 464 468 L 58 M 487 / 475 479G 59 T 499 / 487 491 N 60 P 504 / 492 496 K 61 I 509 / 497 501 L T 499 /487 491 S 62 M 135 / 123 127 Q T 499 / 487 491 V 63 M 135 / 123 127 F T499 / 487 491 V 64 M 135 / 123 127 F T 499 / 487 491 F 65 M 135 / 123127 F T 499 / 487 491 M 66 M 135 / 123 127 F T 499 / 487 491 G

TABLE 2.2 The following mutants were evaluated in the VO1 background(n22-yhcB-t30-VO1, SEQ ID NO: 110) according to wild type SrK0 (SEQ IDNOS: 37 and 108), 8rp-t20SrKO (SEQ ID NOS: 38 and 106), n22yhcB-T30VO1(SEQ ID NO: 104) and n22yhcB- t30VO2 (SEQ ID NOS: 61 and 105). Mutation(relative to SEQ Position Position ID NO: 37 (SEQ SEQ ID NO: 37 (SEQ SEQID NO: 104 / ID NO: 108) / ID NO: 108) / SEQ ID NO: SEQ ID NO: 38 SEQ IDNO: 38(SEQ 105 (SEQ ID (SEQ ID NO: No. WT ID NO: 106) NO: 61) 106) 1 A 2T 2 H 46/34 38 R 3 E 52/40 44 A 4 R 76/64 68 K 5 M 94/82 86 V 6 T131/119 123 K 7 T 131/119 123 Q 8 L 150/138 142 M 9 D 191/179 183 N 10 L231/219 223 M 11 Q 268/256 260 T 12 E 323/311 315 L 13 K 344/332 336 D14 R 351/339 343 Q 15 I 389/377 381 L 16 I 389/377 381 V 17 I 389/377381 A 18 I 390/378 382 L 19 I 390/378 382 M 20 V 400/388 392 Q 21 I444/432 436 A 22 T 468/456 460 I 23 T 488/476 480 D 24 E 491/479 483 K25 I 495/483 487 V

TABLE 2.3 Summary of mutations of several engineered SrKO derivativesbased on alignments relative to wild type SrKO (SEQ ID NOS: 37 and 108).The point mutations for each of the SrKO derivatives (SEQ ID NOS: 38,61, 104, 105, 106, and 107) are identified based on the shift value foreach sequence relative to wild type SrKO. 8rp-t20SrKO (SEQ ID NOS: 38and 106) is the SrKO sequence with a 20-amino acid truncation at theN-terminus, and the addition of an 8-amino acid membrane anchor.8rp-t20VO0 (SEQ ID NO: 107) has a truncation of 20 amino acids of theSrKO N-terminus, the addition of an 8-amino acid N- terminal anchor, anda single mutation at position 487. n22yhcB-t30VO1 (SEQ ID NO: 104) has a30-amino acid truncation of the SrKO N-terminus, a membrane anchor basedon 22 amino acids from E. coli yhcB, and eight point mutations atpositions 38, 223, 276, 375, 392, 436, 480, and 491, and a n22t30-yhcBmutation. n22yhcB-t30VO2 (SEQ ID NOS: 61 and 105) has a 30-amino acidtruncation of the SrKO N-terminus, a membrane anchor based on 22 aminoacids from E. coli yhcB, and nine point mutations at positions 68, 86,123, 223, 276, 375, 382, 460, and 491, and a n22t30-yhcB mutation. WildType 8rp-t20SrKO n22yhcB- SrKO (SEQ (SEQ ID 8rp-t20VO0 n22yhcB- t30VO2(SEQ ID NOS: 37 NOS: 38 and (SEQ ID NO: t30VO1 (SEQ ID NOS: 61 and 108)106) 107) ID NO: 104) and 105) (Shift Value: (Shift Value: (ShiftValue: - (Shift Value: - (Shift Value: - 0) -12) 0) 8) 8) H46 H34 H38RR76 R64 R68K M94 M82 M86V T131 T119 T123Q F231 F219 F223L F223L H284H272 H276Q H276Q R383 R371 R375K R375K 1390 1378 I382L V400 V388 V392Q1444 1432 I436A T468 T456 T460I T488 T476 T480D T499 T487 T487N T491NT491N

The SrKO may be expressed in a variety of host cells, either forrecombinant protein production, or for sesquiterpene (e.g., valencene)oxidation. For example, the host cells include those described in U.S.Pat. No. 8,512,988, which is hereby incorporated by reference in itsentirety. The host cell may be a prokaryotic or eukaryotic cell. In someembodiments the cell is a bacterial cell, such as Escherichia spp.,Streptomyces spp., Zymonas spp., Acetobacter spp., Citrobacter spp.,Synechocystis spp., Rhizobium spp., Clostridium spp., Corynebacteriumspp., Streptococcus spp., Xanthomonas spp., Lactobacillus spp.,Lactococcus spp., Bacillus spp., Alcaligenes spp., Pseudomonas spp.,Aeromonas spp., Azotobacter spp., Comamonas spp., Mycobacterium spp.,Rhodococcus spp., Gluconobacter spp., Ralstonia spp., Acidithiobacillusspp., Microlunatus spp., Geobacter spp., Geobacillus spp., Arthrobacterspp., Flavobacterium spp., Serratia spp., Saccharopolyspora spp.,Thermus spp., Stenotrophomonas spp., Chromobacterium spp., Sinorhizobiumspp., Saccharopolyspora spp., Agrobacterium spp., and Pantoea spp. Thebacterial cell can be a Gram-negative cell such as an Escherichia coli(E. coli) cell, or a Gram-positive cell such as a species of Bacillus.In other embodiments, the cell is a fungal cell such as a yeast cell,such as, for example, Saccharomyces spp., Schizosaccharomyces spp.,Pichia spp., Paffia spp., Kluyveromyces spp., Candida spp., Talaromycesspp., Brettanomyces spp., Pachysolen spp., Debaryomyces spp., Yarrowiaspp., and industrial polyploid yeast strains. In an embodiment, the hostcell is a bacterium selected from E. coli, Bacillus subtillus, orPseudomonas putida. In an embodiment, the host cell is a yeast, and maybe a species of Saccharomyces, Pichia, or Yarrowia, includingSaccharomyces cerevisiae, Pichia pastoris, and Yarrowia lipolytica.

In some embodiments, the host cell produces isopentyl pyrophosphate(IPP), which acts as a substrate for the synthesis of the sesquiterpene.In some embodiments, the IPP is produced by metabolic flux (e.g.,starting with a carbon source supplied to the cell) through anendogenous or heterologous methylerythritol phosphate (MEP) or mevalonicacid (MVA) pathway. In certain embodiments, the MEP or MVA pathway maybe enhanced through expression of heterologous enzymes or duplication ofcertain enzymes in the pathway.

The MEP (2-C-methyl-D-erythritol 4-phosphate) pathway, also called theMEP/DOXP (2-C-methyl-D-erythritol 4-phosphate/1-deoxy-D-xylulose5-phosphate) pathway or the non-mevalonate pathway or the mevalonicacid-independent pathway refers to the pathway that convertsglyceraldehyde-3-phosphate and pyruvate to IPP and DMAPP. The pathwaytypically involves action of the following enzymes:1-deoxy-D-xylulose-5-phosphate synthase (Dxs),1-deoxy-D-xylulose-5-phosphate reductoisomerase (IspC),4-diphosphocytidyl-2-C-methyl-D-erythritol synthase (IspD),4-diphosphocytidyl-2-C-methyl-D-erythritol kinase (IspE),2C-methyl-D-erythritol 2,4-cyclodiphosphate synthase (IspF),1-hydroxy-2-methyl-2-(E)-butenyl 4-diphosphate synthase (IspG), andisopentenyl diphosphate isomerase (IspH). The MEP pathway, and the genesand enzymes that make up the MEP pathway, are described in U.S. Pat. No.8,512,988, which is hereby incorporated by reference in its entirety.For example, genes that make up the MEP pathway include dxs, ispC, ispD,ispE, ispF, ispG, ispH, idi, and ispA. In some embodiments, thesesquiterpene is produced at least in part by metabolic flux through anMEP pathway, and wherein the host cell has at least one additional copyof a dxs, ispD, ispF, and/or idi gene (e.g., dxs and idi; or dxs, ispD,ispF, and/or idi).

The MVA pathway refers to the biosynthetic pathway that convertsacetyl-CoA to IPP. The mevalonate pathway typically comprises enzymesthat catalyze the following steps: (a) condensing two molecules ofacetyl-CoA to acetoacetyl-CoA (e.g., by action of acetoacetyl-CoAthiolase); (b) condensing acetoacetyl-CoA with acetyl-CoA to formhydroxymethylglutaryl-CoenzymeA (HMG-CoA) (e.g., by action of HMG-CoAsynthase (HMGS)); (c) converting HMG-CoA to mevalonate (e.g., by actionof HMG-CoA reductase (HMGR)); (d) phosphorylating mevalonate tomevalonate 5-phosphate (e.g., by action of mevalonate kinase (MK)); (e)converting mevalonate 5-phosphate to mevalonate 5-pyrophosphate (e.g.,by action of phosphomevalonate kinase (PMK)); and (f) convertingmevalonate 5-pyrophosphate to isopentenyl pyrophosphate (e.g., by actionof mevalonate pyrophosphate decarboxylase (MPD)). The MVA pathway, andthe genes and enzymes that make up the MEP pathway, are described inU.S. Pat. No. 7,667,017, which is hereby incorporated by reference inits entirety.

In some embodiments, the host cell expresses a farnesyl pyrophosphatesynthase (FPPS), which produces farnesyl pyrophosphate from IPP orDMAPP. As shown in FIG. 1, farnesyl pyrophosphate is an intermediate forproduction of valencene. An exemplary farnesyl pyrophosphate synthase isERG20 of Saccharomyces cerevisiae (NCBI accession P08524) and E. coliispA. Various other prokaryotic, yeast, plant, and mammalian FPPSenzymes are known, and may be used in accordance with this aspect.

The host cell may further express a heterologous sesquiterpene synthaseto produce the desired sesquiterpene, such as a valencene synthase.Several valencene synthase enzymes are known including valencenesynthase from Citrus x paradisi or from Citrus sinensis. Citrus sinensisVS (e.g., AAQ04608.1) as well as various derivatives thereof aredescribed in US 2012/0246767, which is hereby incorporated by reference.For example, the disclosure may employ an amino acid sequence of Citrussinensis valencene synthase (CsVS) (SEQ ID NO: 12), or a derivative,having from 1 to 30 mutations or from 1 to 20 or from 1 to 10 mutationswith respect to the wild type amino acid sequence (SEQ ID NO: 12). Suchsequences may have at least 60% sequence identity, at least 70% sequenceidentity, at least 80% sequence identity, at least 90% sequenceidentity, at least 95% sequence identity, or at least about 96%, about97%, about 98%, or about 99% sequence identity with the wild typesequence (SEQ ID NO: 12). Further, valencene synthase from Vitisvinifera (VvVS) (SEQ ID NO: 1) has been described by Licker et al.(Phytochemistry (2004) 65: 2649-2659). In an embodiment, a valencenesynthase comprising the amino acid sequence of VvVS (SEQ ID NO: 1) or anengineered derivative thereof may be employed with the presentdisclosure. Various sesquiterpene synthase enzymes such as valencenesynthase are known and are described in, for example, US 2012/0107893,US 2012/0246767, and U.S. Pat. No. 7,273,735, which are herebyincorporated by reference in their entireties.

For example, in some embodiments, the valencene synthase is a VvVSderivative that comprises an amino acid sequence having from about 1 toabout 40 mutations, from about 1 to about 35 mutations, from about 1 toabout 30 mutations, about 1 to about 25 mutations, from about 1 to about20 mutations, about 1 to about 15 mutations, or from about 1 to about 10mutations independently selected from substitutions, deletions, orinsertions with respect to VvVS (SEQ ID NO: 1). For example, the VvVSderivative may comprise an amino acid sequence having at least about 5or at least about 10, but less than about 30 or about 20 mutations withrespect to SEQ ID NO: 1. In various embodiments, the VvVS derivativecomprises an amino acid sequence that has about 1 mutation, about 2mutations, about 3 mutations, about 4 mutations, about 5 mutations,about 6 mutations, about 7 mutations, about 8 mutations, about 9mutations, about 10 mutations, about 11 mutations, about 12 mutations,about 13 mutations, about 14 mutations, about 15 mutations, about 16mutations, about 17 mutations, about 18 mutations, about 19 mutations,about 20 mutations, about 21 mutations, about 22 mutations, about 23mutations, about 24 mutations, about 25 mutations, about 26 mutations,about 27 mutations, about 28 mutations, about 29 mutations, about 30mutations, about 31 mutations, about 32 mutations, about 33 mutations,about 34 mutations, about 35 mutations, about 36 mutations, about 37mutations, about 38 mutations, about 39 mutations, or about 40 mutationsrelative to VvVS (SEQ ID NO: 1). Such sequences may have at least 60%sequence identity, at least 70% sequence identity, at least 80% sequenceidentity, at least 90% sequence identity, at least 95% sequenceidentity, or at least about 96%, about 97%, about 98%, or about 99%sequence identity with SEQ ID NO:1. Exemplary mutations of VvVS areshown in Table 3. Mutations can be guided by a homology model of Vitisvinifera valencene synthase (VvVS) based on the 5-epi-aristolochenesynthase crystal structure as a template (PDB: 5EAT).

TABLE 3.1 Summary of Vitis vinifera valencene synthase mutations withrespect to wild type (SEQ ID NO: 1) No. WT Position Mutation 1 N 23 D 2T 37 R 3 P 38 S 4 V 42 R 5 A 45 E 6 C 46 K 7 Q 50 R 8 K 56 E 9 K 59 R 10R 60 K 11 K 61 M 12 T 63 R 13 T 63 K 14 N 69 Q 15 N 69 K 16 S 71 I 17 Q72 R 18 L 73 K 19 N 75 E 20 F 76 M 21 F 76 L 22 V 80 M 23 V 80 L 24 V 85I 25 A 86 S 26 Q 91 D 27 A 96 I 28 Q 98 E 29 Q 98 D 30 C 101 Y 31 N 102H 32 S 103 D 33 F 104 D 34 F 104 N 35 D 111 E 36 N 116 T 37 I 117 S 38 I117 V 39 I 117 T 40 G 120 R 41 Q 127 H 42 T 130 N 43 I 131 V 44 I 135 V45 T 140 K 46 E 142 K 47 E 148 D 48 A 149 S 49 A 149 D 50 I 151 S 51 R155 K 52 M 157 L 53 G 159 S 54 G 159 N 55 E 162 Q 56 E 162 K 57 A 164 S58 V 168 T 59 G 170 D 60 L 174 M 61 A 175 E 62 A 175 D 63 K 176 E 64 T183 K 65 A 187 S 66 M 188 L 67 E 190 N 68 G 193 K 69 A 201 S 70 N 205 E71 R 206 Q 72 I 208 L 73 R 209 H 74 G 211 R 75 L 212 M 76 E 213 P 77 I221 L 78 V 223 R 79 Q 225 D 80 Q 225 E 81 D 226 K 82 D 226 E 83 A 228 E84 F 229 I 85 H 230 V 86 D 231 N 87 K 232 E 88 T 233 A 89 T 233 V 90 S247 D 91 L 248 M 92 L 248 K 93 K 250 Q 94 E 251 K 95 S 254 K 96 N 255 E97 A 257 S 98 K 261 A 99 E 262 D 100 D 264 G 101 Y 280 F 102 M 283 I 103H 284 M 104 H 284 A 105 G 285 A 106 Y 287 F 107 Q 291 N 108 R 294 L 109R 297 I 110 L 299 M 111 M 305 A 112 M 305 L 113 I 308 M 114 T 318 S 115P 319 L 116 P 319 I 117 K 323 Q 118 R 331 K 119 D 333 E 120 I 334 E 121I 334 V 122 N 335 K 123 N 335 Q 124 N 335 S 125 S 336 A 126 Y 343 W 127Y 348 F 128 V 349 L 129 L 352 I 130 D 353 E 131 D 353 N 132 V 354 T 133Y 355 F 134 K 356 E 135 K 356 N 136 I 358 V 137 E 359 D 138 E 360 Y 139E 363 K 140 E 363 L 141 G 366 A 142 Y 369 N 143 R 370 V 144 V 371 I 145H 372 E 146 H 372 P 147 A 374 G 148 A 374 L 149 E 376 D 150 M 378 I 151N 380 I 152 N 380 K 153 R 383 Q 154 E 394 Q 155 E 394 D 156 E 395 N 157E 395 G 158 H 396 Y 159 H 396 Q 160 E 402 D 161 R 405 E 162 C 414 R 163L 415 M 164 A 417 L 165 T 418 V 166 T 419 H 167 V 422 L 168 M 424 V 169A 428 V 170 T 429 S 171 T 437 F 172 S 438 G 173 D 439 Y 174 K 441 R 175I 442 M 176 I 442 L 177 M 443 V 178 S 444 R 179 N 447 S 180 F 448 T 181M 453 A 182 G 466 E 183 T 469 A 184 Q 478 E 185 Y 479 F 186 G 480 A 187V 481 A 188 S 482 T 189 Y 487 C 190 S 488 E 191 E 489 H 192 F 490 I 193F 490 L 194 Q 491 K 195 Q 491 N 196 Q 493 L 197 I 494 M 198 N 496 D 199D 500 E 200 L 506 M 201 T 509 S 202 V 511 M 203 S 512 P 204 S 512 T 205M 513 K 206 P 514 D 207 L 519 A 208 D 527 E 209 V 528 F 210 E 532 D 211Q 533 E 212 Q 533 G 213 S 535 G 214 V 539 S 215 V 542 L 216 V 542 T 217M 543 I 218 N 546 H 219 V 550 L 220 F 551 L 221 I 552 V 222 N 553 D 223N 553 E 224 A 554 P 225 V 555 I

TABLE 3.2 Summary of mutations evaluated in the Vv2M5 background (SEQ IDNO: 9). No. WT Position Mutation 226 N 18 V 227 V 21 S 228 N 23 D 229 N27 S 230 Q 32 H 231 I 34 L 232 T 35 S 233 T 37 S 234 K 41 S 235 V 42 E236 A 45 E 237 K 46 C 238 K 47 M 239 Q 50 R 240 I 51 V 241 D 53 E 242 K56 E 243 V 67 A 244 A 68 N 245 N 69 D 246 N 69 Q 247 S 71 L 248 Q 72 R249 V 80 I 250 A 86 S 251 Q 91 K 252 C 101 Y 253 N 102 D 254 N 102 H 255M 110 D 256 D 111 E 257 G 112 D 258 I 117 S 259 T 130 N 260 R 143 E 261R 145 N 262 A 149 S 263 S 152 N 264 G 159 N 265 G 159 S 266 V 168 T 267K 176 E 268 K 186 E 269 A 187 S 270 S 191 H 271 Y 194 P 272 H 195 P 273N 205 E 274 L 212 M 275 E 213 P 276 W 219 H 277 V 223 I 278 D 226 E 279A 228 E 280 F 229 S 281 T 233 V 282 V 245 L 283 L 248 M 284 L 256 I 285K 261 A 286 E 262 D 287 C 347 F 288 E 363 A 289 H 372 E 290 V 377 A 291E 395 G 292 E 395 N 293 H 396 Y 294 A 399 T 295 C 414 R 296 E 426 D 297S 438 G 298 M 443 I 299 T 469 A 300 S 488 E 301 K 491 R 302 M 513 K 303A 517 E 304 L 519 V 305 E 532 D 306 V 550 L 307 N 553 D

TABLE 3.3 Summary of mutations evaluated in VS2 background (SEQ ID NO:11). No. WT Position Mutation 308 P 20 R 309 N 23 D 310 I 28 F 311 K 41P 312 K 41 S 313 V 42 D 314 R 44 H 315 Q 50 D 316 Q 50 R 317 E 52 R 318K 61 M 319 N 69 Q 320 Q 72 R 321 L 73 K 322 A 79 I 323 A 86 S 324 H 88 L325 Q 91 H 326 A 96 I 327 Q 98 R 328 C 101 Y 329 N 102 H 330 C 107 F 331I 117 S 332 G 120 L 333 T 140 K 334 R 145 N 335 S 152 V 336 V 154 I 337V 154 P 338 R 155 K 339 M 157 L 340 G 159 M 341 A 175 D 342 K 176 E 343A 177 P 344 L 178 I 345 H 184 Y 346 H 184 Q 347 A 187 S 348 S 191 H 349H 195 N 350 L 196 P 351 A 201 R 352 L 212 M 353 E 213 P 354 A 217 Q 355A 228 E 356 D 231 N 357 K 232 P 358 T 233 V 359 E 236 D 360 D 241 E 361N 255 D 362 A 257 M 363 L 276 P 364 Y 280 F 365 M 283 I 366 V 286 A 367T 300 M 368 T 300 I 369 T 306 L 370 T 306 I 371 L 309 I 372 A 315 V 373E 320 D 374 K 323 R 375 S 336 T 376 E 342 D 377 C 347 L 378 A 350 I 379N 356 H 380 E 363 G 381 Q 368 P 382 N 380 D 383 Q 381 L 384 E 395 G 385A 407 G 386 A 407 S 387 C 414 P 388 A 417 I 389 A 432 I 390 V 436 L 391I 442 P 392 I 442 L 393 S 445 R 394 S 446 M 395 T 450 C 396 S 458 T 397H 459 Y 398 H 459 M 399 H 467 Q 400 T 469 A 401 E 484 P 402 Q 485 H 403Q 485 E 404 V 486 A 405 Y 487 L 406 S 488 E 407 I 494 V 408 N 496 D 409N 496 K 410 M 513 T 411 T 523 I 412 D 527 L 413 I 529 L 414 I 529 M 415E 532 H 416 E 532 Y 417 S 535 A 418 R 544 H 419 N 546 Y 420 N 546 F 421A 548 I 422 V 550 L 423 V 555 I

Thus, in various embodiments, the engineered VvVS may have at leastabout 1 mutation, about 2 mutations, about 3 mutations, about 4mutations, about 5 mutations, about 6 mutations, about 7 mutations,about 8 mutations, about 9 mutations, about 10 mutations, about 11mutations, about 12 mutations, about 13 mutations, about 14 mutations,about 15 mutations, about 16 mutations, about 17 mutations, about 18mutations, about 19 mutations, about 20 mutations, about 21 mutations,about 22 mutations, about 23 mutations, about 24 mutations, about 25mutations, about 26 mutations, about 27 mutations, about 28 mutations,about 29 mutations, about 30 mutations, about 31 mutations, about 32mutations, about 33 mutations, about 34 mutations, about 35 mutations,about 36 mutations, about 37 mutations, about 38 mutations, about 39mutations, or about 40 mutations selected from Table 3. Exemplaryrecombinant valencene synthases Vv1M1 (SEQ ID NO: 3), Vv2M1 (SEQ ID NO:5), Vv1M5 (SEQ ID NO: 7), Vv2M5 (SEQ ID NO: 9), and VS2 (SEQ ID NO: 11)are further depicted in FIG. 3, including an alignment in FIG. 3B. Afurther exemplary recombinant valencene synthase VS3 (SEQ ID NO: 129) isalso depicted herein.

In certain aspects, the disclosure provides polynucleotides comprising anucleotide sequence encoding a valencene synthase modified for increasedexpression of valencene as described above. Such polynucleotides may beexpressed in host cells, either on extrachromosomal elements such asplasmids, or may be chromosomally integrated.

In various embodiments, the SrKO is expressed alongside a P450 reductaseto regenerate the enzyme, or alternatively, the SrKO or derivative isexpressed with the P450 reductase as a chimeric P450 enzyme. Functionalexpression of cytochrome P450 has been considered challenging due to theinherent limitations of bacterial platforms, such as the absence ofelectron transfer machinery and cytochrome P450 reductases, andtranslational incompatibility of the membrane signal modules of P450enzymes due to the lack of an endoplasmic reticulum.

Accordingly, in some embodiments the SrKO is expressed as a fusionprotein with a cytochrome P450 reductase partner. Cytochrome P450reductase is a membrane protein found in the endoplasmic reticulum. Itcatalyzes pyridine nucleotide dehydration and electron transfer tomembrane bound cytochrome P450s. Isozymes of similar structure are foundin humans, plants, other mammals, and insects. Exemplary P450 reductasepartners include, for example, Stevia rebaudiana (Sr)CPR (SEQ ID NOS: 62and 63), Stevia rebaudiana (Sr)CPR1 (SEQ ID NOS: 76 and 77), Arabidopsisthaliana (At)CPR (SEQ ID NOS: 64 and 65), Taxus cuspidata (Tc) CPR (SEQID NOS: 66 and 67), Artemisia annua (Aa)CPR (SEQ ID NOS: 68 and 69),Arabidopsis thaliana (At)CPR1 (SEQ ID NOS: 70 and 71), Arabidopsisthaliana (At)CPR2 (SEQ ID NOS: 72 and 73), Arabidopsis thaliana (At)R2(SEQ ID NOS: 74 and 75); Stevia rebaudiana (Sr)CPR2 (SEQ ID NOS: 78 and79); Stevia rebaudiana (Sr)CPR3 (SEQ ID NOS: 80 and 81); Pelargoniumgraveolens (Pg)CPR (SEQ ID NO: 82 and 83). Any of these P450s can bederivatized in some embodiments, for example, to introduce from 1 toabout 20 mutations, or from about 1 to about 10 mutations. FIG. 6B showsan alignment of amino acid sequences for Arabidopsis thaliana andArtemisia annua CPR sequences (SEQ ID NOS: 72, 74, 68, 64, and 70). FIG.6C shows an alignment of Stevia rebaudiana CPR sequences (SEQ ID NOS:78, 80, 62, and 76). FIG. 6D shows an alignment of eight CPR amino acidsequences (SEQ ID NOS: 74, 72, 82, 68, 80, 62, 78, and 76).

Engineering of P450 fusion proteins is disclosed, for example, in US2012/0107893 and US 2012/0164678, both of which are hereby incorporatedby reference in their entireties. In certain embodiments, the SrKO isfused to the cytochrome P450 reductase partner through a linker.Exemplary linker sequences, which are predominantly serine, glycine,and/or alanine, and optionally from one to five charged amino acids suchas lysine or arginine, include, for example, GSG, GSGGGGS (SEQ ID NO:113), GSGEAAAK (SEQ ID NO: 114), GSGEAAAKEAAAK (SEQ ID NO: 115),GSGMGSSSN (SEQ ID NO: 116), and GSTGS (SEQ ID NO: 117). The linker isgenerally flexible, and contains no more than one, two, or threehydrophobic residues, and is generally from three to fifty amino acidsin length, such as from three to twenty amino acids in length. In otherembodiments, a P450 reductase is expressed in the host cell separately,and may be expressed in the same operon as the SrKO in some embodiments.In some embodiments, the P450 reductase enzyme is expressed separatelyin the host cell, and the gene is optionally integrated into the genomeor expressed from a plasmid.

In certain embodiments the N-terminus of the P450 enzymes may beengineered to increase their functional expression. The N-terminus ofmembrane-bound P450 plays important roles in enzyme expression, membraneassociation and substrate access. It has been reported that the use ofrare codons in the N-terminus of P450 significantly improved theexpression level of P450. Further, since most plant P450 enzymes aremembrane-bound and hydrophobic substrates are thought to enter theenzymes through channels dynamically established between the P450 andmembrane, N-terminal engineering can affect the association of themembrane and P450 and therefore the access of substrate to the enzyme.Accordingly, in an embodiment, N-terminal engineering of SrKO generatesan SrKO derivative that either maintains or shows enhanced valenceneoxidase activity in a host system such as E. coli or yeast. An exemplaryN-terminal sequence is 8rp or MALLLAVF (SEQ ID NO: 112), and otherexemplary sequences include sequences of from four to twenty amino acids(such as from four to fifteen amino acids, or from four to ten aminoacids, or about eight amino acids) that are predominately hydrophobic,for example, constructed predominately of (at least 50%, or at least75%) amino acids selected from leucine, valine, alanine, isoleucine, andphenylalanine.

In some embodiments, the SrKO is a derivative having a deletion of atleast a portion of its N-terminal transmembrane region, and the additionof an inner membrane transmembrane domain from E. coli yhcB orderivative thereof. In these embodiments, the P450 enzyme has a morestable and/or productive association with the E. coli inner membrane,which reduces cell stress otherwise induced by the expression of amembrane-associated P450 enzyme. In some embodiments, the SrKO is aderivative having a deletion of from 15 to 35 amino acids of itsN-terminal transmembrane domain, and the addition of from 15 to 25 aminoacids of the transmembrane domain from E. coli yhcB or derivativethereof. In some embodiments, the N-terminal transmembrane domain of thederivative comprises the amino acid sequence MAWEYALIGLVVGIIIGAVA (SEQID NO: 118), or an amino acid sequence having from 1 to 10 or from 1 to5 amino acid mutations with respect to SEQ ID NO: 118.

In some embodiments, the host cell further expresses one or moreenzymes, such as an alcohol dehydrogenase (ADH). In certain embodiments,the host cell may express an ADH enzyme producing nootkatone fromnootkatol, examples of which include Rhodococcus erythropolis CDH (SEQID NO: 84), Citrus sinensis DH (SEQ ID NO: 86), Citrus sinensis DH1 (SEQID NO: 88), Citrus sinensis DH2 (SEQ ID NO: 90), Citrus sinensis DH3(SEQ ID NO: 92), Vitis vinifera DH (SEQ ID NO: 94), Vitis vinifera DH1(SEQ ID NO: 96), Citrus sinensis ABA2 (SEQ ID NO: 98), Brachypodiumdistachyon DH (SEQ ID NO: 100), and Zingiber zerumbet SDR (SEQ ID NO:102). The ADH may comprise an amino acid sequence having at least 70%,at least 80%, or at least 90% sequence identity to one or more of theenzymes described in this paragraph, and with the activity of convertingnootkatol to nootkatone.

Sesquiterpenes (e.g., valencene and its oxygenated products) can beproduced as biosynthetic products of the non-mevalonate pathway in E.coli comprising two modules: the native upstream pathway formingIsopentenyl Pyrophosphate (IPP) and a heterologous downstreamterpenoid-forming pathway. A multivariate-modular approach to metabolicpathway engineering can be employed to optimize the production ofsesquiterpenes in an engineered E. coli. The multivariate-modularpathway engineering approach is based on a systematic multivariatesearch to identify conditions that optimally balance the two pathwaymodules to minimize accumulation of inhibitory intermediates and fluxdiversion to side products.

WO 2011/060057, US 2011/0189717, US 2012/107893, and U.S. Pat. No.8,512,988 (each of which are hereby incorporated by reference) describemethods and compositions for optimizing production of terpenoids incells by controlling expression of genes or proteins participating in anupstream pathway and a downstream pathway. This can be achieved bygrouping the enzyme pathways into two modules: an upstream (MEP) pathwaymodule (e.g., containing one or more genes of the MEP pathway) and adownstream, heterologous pathway to sesquiterpene production. Using thisbasic configuration, parameters such as the effect of plasmid copynumber on cell physiology, gene order and promoter strength in anexpression cassette, and chromosomal integration are evaluated withrespect to their effect on terpene and terpenoid (e.g., sesquiterpene)production. Expression of genes within the MEP pathway can thus beregulated in a modular method. As used herein, regulation by a modularmethod refers to regulation of multiple genes together. By way ofexample, multiple genes within the MEP pathway can be recombinantlyexpressed on a contiguous region of DNA, such as an operon. It should beappreciated that modules of genes within the MEP pathway, consistentwith aspects of the disclosure, can contain any of the genes within theMEP pathway, in any order. In some embodiments, a gene within the MEPpathway is one of the following: dxs, ispC, ispD, ispE, ispF, ispG,ispH, idi, ispA or ispB. A non-limiting example of a module of geneswithin the MEP pathway is a module containing the genes dxs, idi, ispDand ispF, and referred to as dxs-idi-ispDF.

The manipulation of the expression of genes and/or proteins, includingmodules such as the dxs-idi-ispDF operon, and a FPPS-VS operon, can beachieved through methods known to one of ordinary skill in the art. Forexample, expression of the genes or operons can be regulated throughselection of promoters, such as inducible promoters, with differentstrengths. Several non-limiting examples of promoters include Trc, T5and T7. Additionally, expression of genes or operons can be regulatedthrough manipulation of the copy number of the gene or operon in thecell.

The expression of one or more genes and/or proteins within the MEPpathway can be upregulated and/or downregulated. In certain embodiments,upregulation of one or more genes and/or proteins within the MEP pathwaycan be combined with downregulation of one or more genes and/or proteinswithin the MEP pathway. By way of example, in some embodiments, a cellthat overexpresses one or more components of the non-mevalonate (MEP)pathway is used, at least in part, to amplify isopentyl diphosphate(IPP) and dimethylallyl diphosphate (DMAPP), substrates of GGPPS. Insome embodiments, overexpression of one or more components of thenon-mevalonate (MEP) pathway is achieved by increasing the copy numberof one or more components of the non-mevalonate (MEP) pathway. In thisregards, copy numbers of components at rate-limiting steps in the MEPpathway such as (dxs, ispD, ispF, idi) can be amplified, such as byadditional episomal expression.

In some embodiments, the production of indole is used as a surrogatemarker for sesquiterpene production, and/or the accumulation of indolein the culture is controlled to increase sesquiterpene production. Forexample, in various embodiments, accumulation of indole in the cultureis controlled to below about 100 mg/L, or below about 75 mg/L, or belowabout 50 mg/L, or below about 25 mg/L, or below about 10 mg/L. Theaccumulation of indole can be controlled by balancing protein expressionand activity using the multivariate modular approach described above,and/or is controlled by chemical means.

In other aspects, the disclosure provides a method for making a productcontaining an oxygenated sesquiterpene (as described), which comprisesincorporating the oxygenated sesquiterpene prepared and recoveredaccording to the method described above into a consumer or industrialproduct. For example, the product may be a flavor product, a fragranceproduct, a cosmetic, a cleaning product, a detergent or soap, or a pestcontrol product (e.g., an insect repellant). In some embodiments, theoxygenated product recovered and optionally enriched by fractionation isnootkatol (e.g., a and R nootkatol) and/or nootkatone, and the productis a flavor product selected from a beverage, a chewing gum, a candy, ora flavor additive, or is a pest control product (e.g., an insectrepellant).

The oxygenated product can be recovered by any suitable process,including partitioning the desired product into an organic phase. Theproduction of the desired product can be determined and/or quantified,for example, by gas chromatography (e.g., GC-MS). The desired productcan be produced in batch or continuous bioreactor systems. Production ofproduct, recovery, and/or analysis of the product can be done asdescribed in US 2012/0246767, which is hereby incorporated by referencein its entirety. For example, in some embodiments, oxygenated oil isextracted from aqueous reaction medium, which may be done by using anorganic solvent, such as an alkane such as heptane, followed byfractional distillation. Sesquiterpene and sesquiterpenoid components offractions may be measured quantitatively by GC/MS, followed by blendingof the fractions to generate a desired nootkatone-containing ingredientfor flavour (or other) applications.

In other aspects, the disclosure provides polynucleotides comprising anucleotide sequence encoding a P450 derivative described herein. Thepolynucleotide may be codon optimized for expression in E. coli or yeastin some embodiments. In another example, the polynucleotide may comprisea nucleotide sequence encoding a SrKO fusion protein, optionally with aP450 reductase partner as described herein. In other embodiments, thedisclosure provides polynucleotides comprising a nucleotide sequenceencoding a sesquiterpene synthase variant described herein, which maylikewise be codon optimized for expression in E. coli or yeast. Suchpolynucleotides may further comprise, in addition to sequences encodingthe P450 or sesquiterpene synthase, one or more expression controlelements. For example, the polynucleotide may comprise one or morepromoters or transcriptional enhancers, ribosomal binding sites,transcription termination signals, and polyadenylation signals, asexpression control elements. The polynucleotide may be inserted withinany suitable vector, including an expression vector, and which may becontained within any suitable host cell for expression. Thepolynucleotide may be designed for introduction and/or proteinexpression in any suitable host cell, including bacterial cells andyeast cells, and may be expressed from a plasmid, or may bechromosomally integrated. In some embodiments, the recombinant nucleicacid molecule encodes an SrKO derivative with a higher activity foroxidation of valencene than the wild type enzyme (SEQ ID NO: 37), andhaving a leader sequence as described, such as the leader sequenceMALLLAVF (SEQ ID NO: 112) or leader sequence derived from E. coli yhcB.In certain embodiments, the recombinant nucleic acid molecules furtherencodes either as an operon or as a fusion in frame with the SrKOderivative, an SrCPR or derivative thereof capable of regenerating theSrKO enzyme. When present as a fusion protein, the SrKO derivative andthe SrCPR may be connected by a linking sequence of from 3 to 10 aminoacids (e.g., 5 amino acids). In some embodiments, the linking sequenceis predominately glycine, serine, and/or alanine and may comprise thesequence GSTGS.

In other aspects, the disclosure provides host cells producing anoxygenated sesquiterpene as described herein, and which express all ofthe enzyme components for producing the desired oxygenated sesquiterpenefrom isopentyl pyrophosphate (IPP). For example, the host cell invarious embodiments expresses a farnesyl pyrophosphate synthase, asesquiterpene synthase, and the SrKO or derivative thereof. IPP may beproduced through the MEP and/or MVA pathway, which may be endogenous tothe host cell or modified through expression of heterologous enzymes orduplication of certain enzymes in the pathway. Host cells includevarious bacteria and yeast as described herein.

In still other aspects, the disclosure provides sesquiterpene productsproduced by the methods and host cells described herein. As disclosedherein, SrKO enzyme showed unique activities by creating differentstereoisomers of the hydroxylated product (alpha and beta nootkatol andfurther oxidizing to ketone, nootkatone), and produced differentoxygenated terpene products including hydroxygermacra-1(10)5-diene, andmurolan-3,9(11) diene-10-peroxy. This activity provides for theincorporation of a unique valencene oxidation profile into an oilsuitable for flavouring applications.

In certain embodiments, the processes and methods disclosed hereinprovide compositions and formulations comprising an oxygenated product.In some embodiments, said compositions and formulations may furthercomprise an unoxygenated product, a sesquiterpene, valencene, anon-sesquiterpene component, and/or one or more additional ingredients.In particular embodiments, the compositions and formulations disclosedherein comprise at least one of valencene, hydroxygermacra-1(10)5-diene,murolan-3,9(11) diene-10-peroxy, α-nootkatol, β-nootkatol, andnootkatone. In certain embodiments of the compositions and formulationsdisclosed herein, the compositions and formulations comprise at leastone of nootkatone, α-nootkatol, β-nootkatol, and valencene. For example,in certain such embodiments, the nootkatone content may be selected fromabout 50% to about 65% (w/w), about 52.5% to about 62.5% (w/w), andabout 55% to about 60% (w/w); the α-nootkatol content may be selectedfrom about 15% to about 30% (w/w), about 17.5% to about 27.5% (w/w), andabout 20% to about 25% (w/w); the β-nootkatol content may be selectedfrom about 1% to about 15% (w/w), about 3% to about 12% (w/w), and about5% to about 10% (w/w); and the valencene content may be selected fromabout 1% to about 15% (w/w), about 3% to about 12% (w/w), and about 5%to about 10% (w/w).

In specific embodiments of the compositions and formulations disclosedherein, the compositions and formulations comprise at least one ofnootkatone, α-nootkatol, and β-nootkatol. For example, in certain suchembodiments, the nootkatone content may be selected from about 50% toabout 65% (w/w), about 52.5% to about 62.5% (w/w), and about 55% toabout 60% (w/w); the α-nootkatol content may be selected from about 15%to about 30% (w/w), about 17.5% to about 27.5% (w/w), and about 20% toabout 25% (w/w); and the 3-nootkatol content may be selected from about1% to about 15% (w/w), about 3% to about 12% (w/w), and about 5% toabout 10% (w/w).

In one embodiment, the composition or formulation comprise nootkatone,α-nootkatol, and β-nootkatol, wherein the nootkatone is present in anamount ranging from about 55% to about 60% (w/w), the α-nootkatol ispresent in an amount ranging from about 20% to about 25% (w/w), and theβ-nootkatol is present in an amount ranging from about 5% to about 10%(w/w).

In another embodiment, the composition or formulation comprisevalencene, nootkatone, α-nootkatol, and β-nootkatol, wherein thevalencene is present in an amount ranging from about 5% to about 10%(w/w), the nootkatone is present in an amount ranging from about 55% toabout 60% (w/w), the α-nootkatol is present in an amount ranging fromabout 20% to about 25% (w/w), and the β-nootkatol is present in anamount ranging from about 5% to about 10% (w/w).

Further, other P450 enzymes tested, including previously knownsesquiterpene CYP450's or P450's having hydroxylating activity on thevalencene substrate produced one of the stereoisomers (beta nootkatol)and only minor amounts of the ketone (nootkatone). Specifically, theother sesquiterpene CYP450 enzymes produced beta-nootkatol and hydroxylvalencene as major products, while Taxol CYP450 enzyme did not produceany oxygenated valencene (Table 4 and FIG. 7). The different blend ofsesquiterpene products produced by SrKO provides a unique profile with aunique sensory/taste profile.

In certain aspects, the disclosure relates to SrKO derivative enzymes.In certain embodiments, the SrKO derivative polypeptide comprises anamino acid sequence that has up to 25 mutations compared to the wildtype protein according to SEQ ID NO: 37. For example, the SrKOderivative may comprise an amino acid sequence that has one or moremutations at positions selected from 46, 76, 94, 131, 231, 284, 383,390, 400, 444, 468, 488 and 499, numbered according to SEQ ID NO: 37.For example, in some embodiments, the SrKO is a derivative comprising anamino acid sequence having one or more (or all) of the mutationsselected from H46R, R76K, M94V, T131Q, F231L, H284Q, R383K, I390L,V400Q, I444A, T468I, T488D, and T499N, numbered according to SEQ IDNO:37. In certain embodiments, the SrKO is a derivative comprising anamino acid sequence having one or more (or all) of the mutationsselected from R76K, M94V, T131Q, F231L, H284Q, R383K, I390L, T468I, andT499N, numbered according to SEQ ID NO: 37. In some embodiments, theSrKO derivative comprises an amino acid sequence selected from SEQ IDNOS: 55-61, which were engineered according to this disclosure toimprove activity for oxygenation of valencene (e.g., production ofnootkatone and/or nootkatol). In some embodiments, the derivativecomprises an amino acid sequence having from one to twenty mutationsrelative to a sequence selected from SEQ ID NOS: 55-61, with the provisothat the amino acid sequence has one or more mutations at positionsselected from 46, 76, 94, 131, 231, 284, 383, 390, 400, 444, 468, 488and 499 (numbered according to SEQ ID NO: 37), or the proviso that theSrKO derivative comprises an amino acid sequence having one or more (orall) of the mutations selected from H46R, R76K, M94V, T131Q, F231L,H284Q, R383K, I390L, V400Q, I444A, T468I, T488D, and T499N (numberedaccording to SEQ ID NO: 37). In certain embodiments, the derivativecomprises an amino acid sequence having from one to twenty mutationsrelative to a sequence selected from SEQ ID NOS: 55-61, with the provisothat the amino acid sequence has one or more mutations at positionsselected from 46, 76, 94, 131, 231, 284, 383, 390, 400, 444, 468, 488and 499 (numbered according to SEQ ID NO: 37), or the proviso that theSrKO derivative comprises an amino acid sequence having one or more (orall) of the mutations selected from R76K, M94V, T131Q, F231L, H284Q,R383K, I390L, T468I, and T499N (numbered according to SEQ ID NO: 37). Asshown herein, these mutations increase the level of SrKOs valenceneoxidation activity.

In these or other embodiments, the SrKO is a derivative having adeletion of at least a portion of its N-terminal transmembrane region,and the addition of an inner membrane transmembrane domain from E. coliyhcB or derivative thereof. In some embodiments, the SrKO is aderivative having a deletion of from 15 to 35 amino acids of itsN-terminal transmembrane domain, and the addition of from 15 to 25 aminoacids of the transmembrane domain from E. coli yhcB or derivativethereof. In some embodiments, the N-terminal transmembrane domain of thederivative comprises the amino acid sequence MAWEYALIGLVVGIIIGAVA (SEQID NO:118), or an amino acid sequence having from 1 to 10 or from 1 to 5amino acid mutations with respect to SEQ ID NO:118.

In still other aspects, the disclosure provides a method of preparingthe modified SrKO polypeptide, wherein the method comprises the stepsof: (i) culturing a host cell expressing the modified polypeptide underconditions which permit expression of the polypeptide; and (ii)optionally recovering the polypeptide.

In still other aspects, the disclosure provides a method of producing anoxygenated sesquiterpene comprising the steps of: (i) providing themodified SrKO polypeptide, (ii) contacting a sesquiterpene with themodified SrKO polypeptide, and (iii) recovering the produced oxygenatedsesquiterpene. The method may further comprise providing a CPR enzymefor regenerating the SrKO cofactor (e.g., SrCPR). In some embodiments,the oxygenated sesquiterpene is recovered as an oil. In someembodiments, the sesquiterpene is valencene. In some embodiments, theoxygenated sesquiterpene comprises hydroxygermacra-1(10)5-diene,murolan-3,9(11) diene-10-peroxy, alpha-nootkatol, beta-nootkatol, andnootkatone. In some embodiments, the predominant oxygenated product isnootkatone and/or nootkatol. In some embodiments, the oxygenated productcomprises both alpha and beta nootkatol.

In another aspect, there is provided an SrKO crystal model structure(CMS) based on the structural coordinates of P45017A1, with an aminoacid sequence of SrKO or derivative described herein. The CMS comprisesa terpene binding pocket domain (TBD) that comprises a terpene bindingpocket (TBP) and a terpene (e.g., valencene) bound to the TBD. FIGS. 8Aand 8B. This SrKO crystal model structure (CMS) facilitates in-silicotesting of SrKO derivatives.

Thus, in still other embodiments, the disclosure provides a method ofscreening for a terpene capable of binding to a TBD wherein the methodcomprises the use of the SrKO CMS. In another aspect, the disclosureprovides a method for screening for a terpene capable of binding to theTBP, and the method comprises contacting the TBP with a test compound,and determining if said test compound binds to said TBP. In someembodiments, the method is to screen for a test compound (e.g.,terpenes) useful in modulating the activity of a SrKO enzyme.

In another aspect, the disclosure provides a method for predicting,simulating or modelling the molecular characteristics and/or molecularinteractions of a terpene binding domain (TBD) comprising the use of acomputer model, said computer model comprising, using or depicting thestructural coordinates of a terpene binding domain as defined above toprovide an image of said ligand binding domain and to optionally displaysaid image.

EXAMPLES Example 1: Construction of Sesquiterpene Precursor (Valencene)Producing E. coli Strain

E. coli overexpressing upstream MEP pathway genes dxs, ispD, ispF, andidi was created, which facilitates flux to the isoprenoid precursorisopentyl-pyrophosphate (IPP) supporting more than 1 g/L titers of aheterologous diterpenoid product (3). Strains were constructed producinga variety of terpenoids including mono- and sesquiterpenes by replacingthe geranylgeranyl pyrophosphate synthase (GGPS) and diterpene synthasewith a farnesyl pyrophosphate synthase (FPPS) and sesquiterpene synthaseor a geranyl pyrophosphate synthase (GPPS) and monoterpene synthase. Fordeveloping a sesquiterpene producing strain to test the CYP450s fornovel oxygenated terpenes, a valencene synthase enzyme was cloned andexpressed in the MEP pathway overexpressed E. coli strain. The highsubstrate flux helps identify the activity of the CYP450. Previously,research on an oxygenated taxadiene producing strain showed asignificant drop in the productivity upon transferring the CYP450pathway to the taxadiene producing strain (300 mg/L to ˜10 mg/L).

Further, multivariate modular metabolic engineering (MMME) was appliedfor balancing the pathway for high level production of valencene.Naturally occurring valencene synthases, such as that from Vitisvinifera, often perform sub-optimally (˜5 mg/L) even after MMMEoptimization, compared to previous results obtaining 100's of mg/Lditerpenoids. Enzymes involved in the sesquiterpene biosynthesis can bedifficult to express in E. coli, and also are deficient in kineticsrelative to those involved in primary metabolism (17).

A homology model for the Vitis vinifera valencene synthase (VvVS) wasconstructed using the BioLuminate® software package (Schrodinger, Inc.)with the 5-epi-aristolochene synthase crystal structure as a template(PDB: 5EAT). Further, to identify the natural mutational landscape ofterpene synthases, an extensive multiple sequence alignmentincorporating hundreds of related terpene synthase sequences wascreated. Using this information, mutations were designed using acombination of back-to-consensus, in silico energetics, and structuralanalysis. Back-to-consensus mutations have been shown to be an importanttool for improving stability (19,20) and expression (21). Energeticscalculations based on atomic force-field models in BioLuminate were usedto assess the ΔΔG of folding for individual mutations predicted forpositions with low solvent-accessible surface area, which were predictedto affect folding and stability.

By applying the MMME approach, a balanced upstream and downstreamvalencene production strain was identified incorporating acodon-optimized version of VvVS on a plasmid with a p15A origin ofreplication and a T7 promoter. This strain background was then used toscreen designed synthase enzyme mutations. Using the aforementionedprotein engineering tools we designed over 200 unique point mutations(Table 3) which were then constructed in the p15A-T7 screening plasmidusing site-directed mutagenesis. Mutated enzyme variants weretransformed into the screening strain, triplicate colonies were culturedin selective LB cell culture medium overnight, and then inoculated intoa minimal R-medium and cultured for four days at 22° C. Cultures wereextracted using methyl tert-butyl ether (MTBE) and analyzed by combinedgas chromatography/mass spectrometry for productivity of valencene.

Approximately one-fifth of the designed point mutations increasedvalencene productivity in our screening strain by at least 20% (FIG. 2).Beneficial point mutations were then strategically combined to conferincreasingly advantageous phenotypes. Recombined valencene synthasesequences are provided as Vv1M1 (Mutations—R331K, I334E, N335S, V371I,A374L, T418V, S482T, S512P, K356N, Q491K, E394D, A428V, Y348F, T318S,L352I, I442L, A554P), Vv2M1 (Mutations—R331K, I334E, N335S, V371I,A374L, T418V, S482T, S512P, K356N, Q491K, E394D, A428V, V542T, G480A,M305L, K441R, A554P), Vv1M5 (Mutations—R331K, I334E, N335S, V371I,A374L, T418V, S482T, S512P, K356N, Q491K, E394D, A428V, Y348F, T318S,L352I, I442L, A554P, H284M, C46K, F448T, Q533E), and Vv2M5(Mutations—R331K, I334E, N335S, V371I, A374L, T418V, S482T, S512P,K356N, Q491K, E394D, A428V, V542T, G480A, M305L, K441R, A554P, H284M,C46K, F448T, Q533E) (FIG. 3). When either of these enzymes wasoverexpressed in our MEP pathway strain with dxs-idi-ispDFoverexpressed, and balanced using MMME, the titers of valencene obtainedwere sufficient to motivate incorporation of P450 enzymes to test theirability to catalyze the formation of oxygenated valencene. Titers ofvalencene before P450 incorporation were about 30 mg/L.

Example 2: Functional Activity of CYP450 Library on Valencene Scaffold

Valencene was used as a model system to validate the power ofCYP450-based oxygenation chemistry for production terpene chemicals.

The CYPP450 candidate screening was conducted using the valenceneproducing E. coli strains as host background. For constructing theCYP450 for functional expression, a proprietary plasmid system, p5Trc(plasmid derived from pSC101) was used to construct a plasmid containingthe candidate P450 fused to an N-terminal truncated Stevia rebaudianacytochrome P450 reductase (SrCPR) through a flexible 5-amino acid linker(GSTGS, SEQ ID NO: 117). The sequences of the various candidate P450sare shown in FIG. 4. The candidate CYP450's were analyzed for N-terminalmembrane associating regions which were truncated and a 8-amino acidleader sequence (MALLLAVF, SEQ ID NO: 112) was added to the fusion(FIGS. 5A and 5B). CPR red/ox partners from Arabidopsis thaliana andTaxus cuspidata were also prepared in similar genetic constructions.Since the native SrCPR was effective, the level of activity of theseconstructs was not determined. The sequences of the various CPR red/oxpartners are shown in FIG. 6. Following transformation ofp5Trc-CYP450-L-SrCPR to valencene producing strain, the strains werecultured overnight at 30° C. in antibiotic selective LB media. Thesecultures were then used to inoculate 2 mL antibiotic selective R-mediacultures in hungate tubes with 15 g/L glycerol and 0.1 mM IPTG whichwere subsequently cultured for 4-days at 22° C. before being extractedwith methyl tert-butyl ether (MTBE).

A set of CYP450 enzymes, from those listed in Table 4, was selected andclassified for both sesqui- and diterpene oxygenation in this E. colisystem. Among the various CYP450 enzymes tested foroxygenation onvalencene, kaurene oxidase from Stevia rebaudiana (SrKO) (16) wasdiscovered to have a unique oxygenation chemistry on the valencenescaffold. SrKO natively oxidizes the diterpene (−)-kaurene at the C19position to (−)-kaurenoic acid. SrKO enzyme showed unique activities inthe present studies by creating different stereoisomers of thehydroxylated product (alpha and beta nootkatol and further oxidizing tothe ketone, nootkatone), and produced different oxygenated terpeneproducts including hydroxygermacra-1(10)5-diene, murolan-3,9(11)diene-10-peroxy, in addition to the alpha-nootkatol, beta-nootkatol, andnootkatone. Other P450's, including the previously known sesquiterpeneCYP45's for hydroxylating valencene produced only one of the isomers(beta nootkatol) and only detectable amounts of ketone (nootkatone).Specifically, the other sesquiterpene CYP450 enzymes producedbeta-nootkatol and hydroxyl valencene as major products, while anotherditerpene CYP45 enzymes (e.g., Taxus 5-alpha hydroxylase) producednootkatol as only a minor (detectable) product (Table 4 and FIG. 7).

TABLE 4 Major Products Formed From Valencene by Select P450 Enzymes inE. coli SPECIES NAME MAJOR PRODUCTS Cichorium CiVO β-nootkatol,α-cadinol, hydroxyl intybus valencene. Hyoscyamus HmPO β-nootkatol,a-cadinol, hydroxyl muticus valencene, nootkatone Latuca LsGAOβ-nootkatol, α-cadinol, isovalencenol, spicata nootkat-11-en-10-ol.Bamadesia BsGAO β-nootkatol, α-cadinol, isovalencenol. spinosa NicotianaNtEAO α-cadinol, nootkat-11-en-10-ol. tabacum Stevia SrKO α-nootkatol,hydroxygermacra-1(10)5- rebaudiana diene, β-nootkatol, nootkatone,murolan-3,9(11) diene-10-peroxy Zingiber ZzHO α-cadinol,nootkat-11-en-10-ol. zerumbet Citrus × CpVO α-cadinol,nootkat-11-en-10-ol. paradisi Mentha MsL6OH α-cadinol. spicata NicotianaNtVO α-cadinol, nootkat-11-en-10-ol. tabacum Solanum StVO α-cadinol,β-nootkatol, globulol. tuberosum Arabidopsis AtKO α-cadinol,nootkat-11-en-10-ol. thaliana Cichorium Ci2VO β-nootkatol, α-cadinol,isovalencenol. intybus Artemesia AaAO α-cadinol, murolol,nootkat-11-en-10- annua ol. Taxus 5-alpha α-cadinol hydroxylase P450

Example 3: Structural and Mutational Studies of SrKO

Once the unique activities of SrKO were identified, experiments wereconducted to improve its ability to conduct its diverse oxidation ofvalencene. The crystal structure for SrKO has not been described. Blastsearch of SrKO against RCSB Protein Data Bank shows the sequenceidentity of SrKO to P450 enzymes with crystal structures are low (˜20%).Given the conservative folding structures of P450s regardless of its lowsequence identity, state-of-the-art protein modeling tools were used tobuild on SrKO. The crystal structure of membrane-bound cytochrome P45017 A1 (see DeVore N. M., Scott E. E., Nature, 482, 116-119, 2012) whichcatalyses the biosynthesis of androgens in human was selected as thetemplate for model development. Using BioLuminate protein modelingsoftware, a homology model was developed (FIG. 8A) such that thepositioning of key residues and characteristic motifs (see Gotoh O., J.Biol Chem, 267, 83-90, 1992) aligned well with the template.Furthermore, a homology model for SrKO which included the prostheticheme-iron complex was also constructed. AutoDock VINA was then used tocreate an ensemble of possible binding modes for valencene in the SrKOactive site (FIG. 8B) (29).

In addition, a Blast search of SrKO against NCBI non-redundant proteinsequence library returned no orthologs with sequence identity greaterthan 80% (except the SrKO itself). The top hits are listed in the Table5.

TABLE 5 BLAST search with SrKO in preparation of Homology Model SequenceEnzyme Name Species Identity Accession kaurene oxidase Stevia rebaudiana99% AAQ63464.1 ent-kaurene oxidase 2 Lactuca sativa 79% BAG71198.1ent-kaurene oxidase 1 Lactuca sativa 71% BAG71197.1 ent-kaurene oxidaseRicinus communis 63% XP_002510288.1

Once the unique activities of SrKO were identified, experiments wereconducted to improve its ability to conduct its diverse oxidation ofvalencene. Using the back-to-consensus mutagenesis strategy, a multiplesequence alignment of P450 enzymes was constructed including sequences(after clustering and elimination of sequences with greater than 90%identity) from a BLAST search ofthe Uniref100 database using 4 seedkaurene oxidase genes, from a BLAST search ofthe bacterial proteomeusing P450_(BM3), P45_(CAM), and P40_(eryF) as seed genes, and the mostclosely related SrKO homologs. Based on the homology model, the multiplesequence alignment, and the literature, various point mutations anddouble mutations were designed and tested. These cytochrome P450derivatives were assessed for improvements in total oxygenated terpeneproductivity (e.g., total of the major peaks observed by GUMS) in the invivo testing system described above. Mutagenesis on active sitepositions guided by the model revealed several variants withsignificantly improved oxygenated products (Table 6 and Table 7 below).

TABLE 6 Binding pocket mutations and their fold productivity of totaloxygenated oil according to wild type SrKO (SEQ ID NOS: 37 and 108),8rp-t20SrKO (SEQ ID NOS: 38 and 106), n22yhcB-t30VO1 (SEQ ID NO: 104)and n22yhcB-t30VO2 (SEQ ID NOS: 61 and 105). Mutants Mutants (numbered(numbered according to Mutants according to SEQ ID NO: 104/ (numberedSEQ ID NO: 37) SEQ ID NO: 105 according to (SEQ ID NO: 108) (SEQ ID NO:61)) Fold Mutant # SEQ ID NO: 38) (Shift value (Shift value productivity(Table (SEQ ID NO: relative to SEQ relative to SEQ (as measured 2.1)106) ID NO: 38: +12) ID NO.: 38: +4) in mg/L) 38 I310V I322V I314V 1.537 I310T I322T I3I4T 0.0 42 V375I V387I V379I 1.4 41 V375T V387T V379T0.0 19 M123F M135F M127F 0.0 20 M123T M135T M127T 0.3 18 M123Q M135QM127Q 0.0 59 T487N T499N T491N 2.5 66 M123F_T487G M135F_T499GM127F_T491G 0.0 63 M123F_T487V M135F_T499V M127F_T491V 0.0 62M123Q_T487V M135Q_T499V M127Q_T491V 0.0 59 T487N_V375F T499N_V387FT491N_V379F 2.2 59 T487N_V375A T499N_V387A T491N_V379F 1.8 59T487N_V121A T499N_V133A T491N_V125A 2.0 59 T487N_V375M T499N_V387MT491N_V379M 1.9 59 T487N_M120L T499N_M132L T491N_M124L 1.8 59T487N_M120I T499N_M132I T491N_M124I 1.8 59 T487N_L114V T499N_L126VT491N_L118V 1.4 59 T487N_F219L T499N_F231L T491N_F223L 3.5 59T487N_M120V T499N_M132V T491N_M124V 1.1 59 T487N_F219I T499N_F231IT491N_F223I 3.3 59 T487N_L114F T499N_L126F T491N_L118F 1.2

TABLE 7 Non-binding pocket point mutations and productivity of totaloxygenated oil compared to the wild type SrKO (SEQ ID NOS: 38 and 106).Mutants Mutants (numbered (numbered according to according to SEQ ID NO:37) SEQ ID NO: 104/ Mutants (SEQ ID NO: 108) SEQ ID NO: 105 (numbered(Shift value (SEQ ID NO: 61)) Fold Mutant # according to relative to(Shift value productivity (Table SEQ ID NOS: SEQ ID NO: relative to SEQ(as measured 2.1) 38 and 106) 38: +12) ID NO: 38: +4) in mg/L) 53 G442AG454A G446A 0.849153 55 L454M L466M L458M 0.717318 44 I378V I390V I382V0.349005 47 V388M V400M V392M 0.792428 9 V85I V97I V89I 0.795913 51V413K V425K V417K 0.902039 60 P492K P504K P496K 0.131657 40 R371I R383IR375I 0.657808 7 T80C T92C T84C 0.342501 23 A140R A152R A144R 0.014872 2Y59H Y71H Y63H 0.406787 5 A67E A78E A71E 0.937429 8 M82V M94V M86V1.585588 11 S86N S98N S90N 0.977752 22 Y129F Y141F Y133F 0.686276 24K149R K161R K153R 0.990776 29 D208E D220E D212E 0.853446 31 S267A S279AS271A 0.79152 32 H272Q H284Q H276Q 0.958227 33 S284C S296C S288C0.652348 39 R371K R383K R375K 1.443497 45 H382Y H394Y H386Y 0.609951 46V388Q V400Q V392Q 0.924043 49 L400I L412I K4041 0.682775 50 V413D V425DV417D 0.039261 52 F434L F446L F438L 0.793926 57 M464L M476L M468L0.689696 58 M475G M487G M479G 0.573906 61 I497L I509L I501L 0.679949 15A116R A128R A120R 0.216353 1 L47I L59I L51I 0.88992 25 H150F H162F H154F0.666723

Example 4: Isolation and Evaluation of Oxygenated Product

The product derived from oxidation of valencene by the cytochrome P450enzyme SrKO (SEQ ID NO: 38) was analysed by GC/MS (Agilent 6800; Column:Rtx-5, 0.32 mm×60 m×1.0 μm film thickness; GC Temp. Program: 40° C. for5 min, increased at 4° C./min to 300° C. and held for 30 min.) resultingin the data provided in Table 8A and 8B.

TABLE 8A SrKO oxidation of valencene Ret. GC-FID Time Compound Name CAS# Area % 33.762 dodecane 112-40-3 6.70 35.440 glyceryl diacetate I 5.2638.767 triacetin 102-76-1 4.48 39.518 unknown 10.17 40.176 unknown 7.5242.012 unknown 2.53 44.437 unknown 20.25 44.816 valencene 4630-07-3 1.9745.546 nootkatene 5090-61-9 1.09 46.260 unknown 2.14 46.395 unknown 6.7746.869 unknown 4.01 47.394 germacrene D-4-ol 74841-87-5 1.23 48.273unknown 1.86 49.659 T-muurolol 19912-62-0 0.69 49.753 an unknownsesquiterpene 0.56 50.336 an unknown sesquiterpene 0.58 51.025epinootkatol (or alpha nootkatol) 50763-66-1 1.96 51.430 Nootkatol (orbeta nootkatol) 50763-67-2 3.54 54.138 nootkatone 4674-50-4 15.87 54.5016-isopropenyl-4,8a-dimethyl- 76784-84-4 0.844a,5,6,7,8,8a-hexahydro-2(1H)- naphthalenone TOTAL 100.00

TABLE 8B SrKO oxidation of valencene Ret. GC-FID Time Compound Name CAS# Area % 33.763 dodecane 112-40-3 7.26 35.470 glyceryl diacetate I 7.0738.773 triacetin 102-76-1 6.19 39.526 unknown 11.56 40.179 unknown 8.1044.440 unknown 23.95 44.821 valencene 4630-07-3 6.88 45.545 nootkatene5090-61-9 2.22 46.404 unknown 5.08 46.879 unknown 3.66 47.399 germacreneD-4-ol 74841-87-5 2.89 48.279 unknown 2.27 49.665 T-muurolol 19912-62-00.94 50.342 an unknown sesquiterpene 1.71 51.027 epinootkatol 50763-66-12.48 51.444 nootkatol 50763-67-2 5.24 54.152 nootkatone 4674-50-4 2.49TOTAL 100.00

Similar analysis was conducted on the product produced by SrKOderivatives. It was confirmed that product profiles are comparable, andthat the major products of nootkatone, α-nootkatol, and β-nootkatol canbe produced at higher levels based on mutagenesis of SrKO.

The oxygenated oil product can then be extracted from the aqueousreaction medium using an appropriate solvent (e.g., heptane) followed byfractional distillation. The chemical composition of each fraction canbe measured quantitatively by GC/MS. Fractions can be blended togenerate the desired alpha/beta nootkatol and/or nootkatone ingredientsfor use in flavour or other applications.

Verification of acceptability can be carried out by direct comparison toa reference nootkatone flavouring product (for example, an existingnatural flavouring commercial product obtained from Frutarom) withanalysis provided in Table 9.

TABLE 9 Analysis of commercially available natural flavouring nootkatonefrom Frutarom Ret. GC-FID Time Compound Name CAS # Area % 42.307limonene glycol 1946-00-5 0.201 42.792 decanoic acid 334-48-5 0.11549.405 valencene 4630-07-3 0.039 50.362 delta-cadinene 483-76-1 0.26852.757 alpha-elemol 639-99-6 2.178 53.11 spathulenol 6750-60-3 0.26453.423 caryophyllene oxide 1139-30-6 0.394 53.748 viridiflorol 552-02-30.061 54.225 unknown sesquiterpenoid 0.113 (MW = 220, tent) 54.853unknown 2.985 55.386 unknown 2.251 55.97 T-muurolol 19912-62-0 0.39956.192 bulnesol 22451-73-6 0.722 56.523 7(11), 4b-selinenol; tentative1.425 56.65 unknown (MW = 232, tent) 0.663 56.937 beta-sinensal3779-62-2 0.914 57.449 unknown 0.285 57.589 cedrenal; tentative 0.43858.189 unknown sesquiterpenoid(s) 1.077 58.73 unknown sesquiterpenoids0.537 (MW = 220, 222, tent) 59.102 beta, gamma-nootkatone 35936-67-51.805 59.32 myristic acid 544-63-8 0.058 59.537 1,10-dihydronootkatone20489-53-6 0.582 59.75 a nootkatone isomer 0.442 60.507 nootkatoneisomers (2); 0.812 tentative 60.782 unknowns (2) 0.605 61.034hexadecanal 629-80-1 0.302 62.93 nootkatone 4674-50-4 74.287 63.0573,11-eudesmadiene-2-one 86917-81-9 1.909 (5S,7R,10R) 63.14 unknown (MW =234, tent) 0.18 63.26 unknown (MW = 232, tent) 0.105 64.112 heptadecanal629-90-3 0.344 64.403 unknown sesquiterpenoid 0.446 65.16 unknownsesquiterpenoid 0.147 65.384 palmitic acid 57-10-3 0.154 65.599alpha-camphorene 532-87-6 0.249 65.75 unknown(s) 0.054 65.878dehydro-alpha-vetivenone; 0.115 tentative 66.056 nootkatone, 9-oxo86925-44-2 0.172 66.371 ethyl palmitate 628-97-7 0.185 66.856cis-9-octadecenal 2423-10-1 0.239 66.986 unknown sesquiterpenoids 0.11467.556 octadecanal 638-66-4 0.096 74.551 osthol 484-12-8 0.367 80.543isomerazin 1088-17-1 0.112 84.671 unknown (MW = 298) 0.07 TOTAL 99.28

Two exemplary methods of verification are: 1) Duo-Trio Test, 2)Forced-Choice Preference Test. In one method, the SrKO derived productcan be compared to the reference product (for example a commercialFrutarom sourced nootkatone ingredient) in a duo-trio test to determineif the ingredients can be distinguished with statistical significance.This test will determine if the two nootkatone containing ingredients atleast match one another based on perception of overall taste and aromaprofiles. In the second test, assuming the two products are determinedto be distinguishable in a duo trio test, one could determine if theSrKO derived nootkatone is preferred by conducting a forced-choicepreference test. More details on these tests are provided as follows.

Duo-Trio Test:

A Duo-Trio Test can be conducted to determine if the blended fractionsobtained from the SrKO derived nootkatone flavouring can bedistinguished with statistical significance from a reference nootkatoneproduct (for example, a commercially available nootkatone flavouringsourced from Frutarom). The test will determine if the nootkatoneflavouring ingredients at least match in terms of overall taste andaroma profile typically conducted in a sugar/acid solution but couldalso be evaluated in water or sugar water.

TABLE 10 Name Finished Beverage Spring Water 1000 g Citric Acid 0.1%Sucrose   8% Nootkatone flavouring 2.5 ppm

Methodology: One ounce of the reference sample, labelled “REF” ispresented first followed by a one ounce sample of the reference and aone ounce sample of the test sample presented blindly in random order toa minimum of 15 discriminator panellists. The panellists are asked whichblind sample is the same as the reference sample. The data are subjectedto a statistical analysis to determine the degree of difference betweenthe test sample and the reference control.

Forced-Choice Preference Test:

Assuming a difference is observed between nootkatone flavouring derivedfrom SrKO oxidation and the reference nootkatone product (for example, aFrutarom nootkatone flavouring), a Forced-Choice Preference Test can beconducted to determine if one sample is preferred over the other as anootkatone flavouring ingredient. The test can be conducted insugar/acid solution, sugar water or water.

TABLE 11 Name Finished Beverage Spring Water 1000 g Citric Acid 0.1%Sucrose   8% Nootkatone flavouring 2.5 ppm

Methodology: One ounce of each test sample is presented blindly inrandom order to a minimum of 40 discriminator panellists. The panellistsare asked which blind sample is preferred based on aroma and taste whenconsumed orally and are forced to make a decision. The data aresubjected to a statistical analysis to determine the degree ofpreference for one sample over the other.

Example 5: N-Terminal Anchor Engineering

To optimize membrane interaction of the initial SrKO variants (referredto in these examples as Valencene Oxidase 1, or VO1), E. coli proteinsanchored in the inner membrane with a cytoplasmic C-terminus wereidentified. An N-terminal sequence of E. coli yhcB was selected, whichprovides a single-pass transmembrane domain. 20-24 amino acids from theN-terminus of yhcB was exchanged for the original membrane anchorsequence MALLLAVF (SEQ ID NO:112), and the size of the SrKO N-terminaltruncation was varied from 28 to 32. See FIG. 9. VO1 was expressed undercontrol of a T7 promoter on a p5 plasmid. SrCPR was expressedindependently from the chromosome. Strains were cultured in 96 deepwellplates at 30° C. for 48 hours, in R-medium plus glycerol and dodecaneoverlay as already described.

As shown in FIG. 10, n20yhcB_t29VO1 exhibited 1.2-fold productivity intotal oxygenated titer compared to the average of controls.N20yhcB_t29VO1 exhibited a total oxygentated titer approximately 1.8fold of the original 8RP anchor (not shown).

Example 6: Mutational Analysis of VO1

Mutational analysis of VO1 was conducted in an effort to increaseoxygenated titers, as well as to produce altered product profiles.Strain MB2509 (MP6-MEP MP1-ScFPPS Fab46-VS2 MP6-ScCPR) was used as thebackground, which when transformed with a p5-T7-yhcB-VO1 plasmidproduces about 18% nootkatone, about 35% α-nootkatol, and about 47%β-nootkatol, with a complete conversion of valencene. Strains wereevaluated for higher production of nootkatone and α-nootkatol.

Guided by the homology model based on P450 17A1 (Example 3)site-saturation mutagenesis of the VO active site was conducted at 18positions, and 5 paired position libraries were constructed. First shellresidues were identified through substrate docking, and non-conservedfirst shell residues were selected based on relative proximity andposition for altering the binding pocket geometry. Paired positionlibraries were constructed by overlap extension PCR and Gibson assembly.

TABLE 12 Paired Position Libraries (numbered according to SEQ ID NOS: 37and 108) Library Pos. 1 Allowed AA Pos. 2 Allowed AA 1 V387 F, L, I, S,P, T, A, M P388 S, T, A 2 M132 F, L, I, V, S, P, T, A V133 F, L, I, S,P, T, A, M 3 L123 F, I, V, S, T, A, P, M L126 F, I, V, S, T, A, P, M 4V387 F, L, I, S, P, T, A, M I322 F, L, V, S, P, A, M, T 5 I322 F, L, V,S, P, A, M, T V133 F, L, I, S, P, T, A, M

Strains were evaluated as in Example 4 for total oxygenation ofvalencene, and ratio of α- to β-nootkatol. Strains were evaluated at 30°C. and 22° C.

Primary screening of paired position libraries revealed that many of thevariants lost activity. Library 3 contained variants with improvedactivity at 22° C. but not 30° C. Thus, introducing two or moremutations simultaneously in the first shell residues can bedetermimental to activity.

TABLE 13 The following single position SSM was conducted (numberedaccording to SEQ ID NOS: 37 and 108) Residue Location I390 Channel L392Channel V387 1st Shell E323 1st Shell I helix I322 1st Shell I helixT499 1st Shell Q500 1st Shell L231 1st Shell F helix L123 1st Shell B-Cloop L126 1st Shell B-C loop V125 1st Shell B-C loop V133 1st Shell F87on BM3 T131 Channel M135 1st Shell L234 1st Shell F helix P238 1st ShellF-G loop M132 1st Shell B-C loop P388 1st Shell

Several variants improved oxygenated titers up to 1.7-fold or improvedα/β-LGN ratios up to 3.8-fold. Mutations at positions E323, 1390, andQ500 showed several hits with improved oxygenation titer and/or improvedα/β profile, and these positions were selected for secondary screening.

Next, back-to-consensusmutations (19 mutants) were screened in the VO1background. Using the screening process described in Example 3, thefollowing mutations were screened: A2T, I389L, I389V, I389A, M94V,T488D, E491K, E52A, H46R, D191N, L150M, I495V, T468I, K344D, Q268T,R351Q, R76K, V400Q, and I444A (numbered according to SEQ ID NO: 37). Asshown in FIG. 13A, more than 50% of the mutations resulted in 1.2 to1.45 times oxygenated titers (shown as mg/L), without dramatic shifts inproduct profile. Improvements were seen with A2T, M94V, T488D, E52A,H46R, L150M, T468I, K344D, Q268T, R351Q, R76K, V400Q, and I444A, whichwere selected for secondary screening. FIG. 13B shows the same screenplotted versus fold total oxygenated product change and α-/β-nootkatolratio.

Lead variants from active site SSM (L231M, I390L, I390M, T131K, andT131Q), the N-terminal anchor variant n20yhcB_t29VO1, andback-to-consensus mutagenesis were selected, and re-screened. Theresults of this secondary screen are shown in FIG. 14. Several mutationsshowed a 1.1-1.4-fold improvement in oxygenated titers. To narrow thelist of mutations for recombination, the same mutations were screened at33° C. to differentiate stabilizing mutations which could enable aprocess shift to higher temperature. As shown in FIG. 15, six mutations(M94V, L150M, T468I, R76K, I390L, and T131Q) maintained improvedproductivities at 33° C. These six mutations, in addition to the leadN-terminal anchor, were selected for recombination.

Example 7: SrKO Recombination Library Screening

The seven mutations selected after secondary screening (Example 6) wererandomly incorporated into a VO recombination library by allowing eitherthe variant or wild type at each site. The background strain was MB2509(EGV G2 MP6-CPR)+pBAC-T7-BCD7-yhcB-VO.

Primary screening at 30° C. (using the same process described in Example5) identified several variants with up to 1.35-fold improvement inoxygenated product titers, compared to VOL. Further, select variantsshowed a shift in production to nootkatone, suggesting higher P450activity (since production of nootkatone requires two oxygenationcycles). Results of primary screening are shown in FIG. 16A (strainversus titer in mg/L). FIG. 16B presents the same screen shown based onoxygenation capacity (total of nootkatone, α-nootkatol, andβ-nootkatol).

The recombination variants were then screened at 34° C. and 37° C. toselect leads with improved activity and stability at higher temperature.The results of the secondary screen are shown in FIG. 17. While thecontrol was almost completely inactive at 37° C., six leads showedpromising activity at the higher temperatures, and were selected forfurther screening (c11(8), b4(7), c6(1), c12(3), b6(2), and c9(6)).Based on this further screening (FIG. 18) c6(1) was selected as the bestvariant based on oxygenation capacity. Variant c6(1) was furthermodified to generate V02 (SEQ ID NO: 111). The six leads contain thefollowing sets of mutations.

TABLE 14 Sets of mutations in lead variants from recombination library(shown relative to SEQ ID NOS: 37 and 108). N20_t29yhcB R76K M94V T131QL150M I390L T468I A39H Others c9(6) X X X V146L c12(3) X X X X b4(7) X XX X X c11(8) X X X X X X c6(1) X X X X X b6(2) X X X

FIG. 23 (A and B) shows alignments of several engineered valenceneoxidase (VO) variants as described herein, and highlights selectmutations evaluated in the screening process. In FIG. 23A: 8rp-t20SrKO(SEQ ID NO: 106) is the SrKO sequence with a 20-amino acid truncation atthe N-terminus, and the addition of an 8-amino acid membrane anchor.8rp-t20VO0 (SEQ ID NO: 107) has a truncation of 20 amino acids of theSrKO N-terminus, the addition of an 8-amino acid N-terminal anchor, anda single mutation at position 499 (numbered according to wild-typeSrKO). n22yhcB-t30VO1 (SEQ ID NO: 104) has a 30-amino acid truncation ofthe SrKO N-terminus, a membrane anchor based on 22 amino acids from E.coli yhcB, and eight point mutations at positions 46, 231, 284, 383,400, 444, 488, and 499 (with respect to SrKO wild-type). n22yhcB-t30VO2(SEQ ID NOS: 61 and 105) has a 30-amino acid truncation of the SrKON-terminus, a membrane anchor based on 22 amino acids from E. coli yhcB,and nine point mutations at positions 76, 94, 131, 231, 284, 383, 390,468, and 499 (with respect to SrKO wild-type). In FIG. 23B, pointmutations in VO0 (SEQ ID NO: 109), VO1 (SEQ ID NO: 110), and VO2 (SEQ IDNO: 111) are shown against wild-type SrKO (SEQ ID NO: 108) (all shownwith the wild-type SrKO N-terminus for convenience).

Example 8: Cytochrome P450 Reductase Screening

A set of cytochrome P450 reductases were screened for improved activitywith VO1. This example was done using the strain MB2459 as thebackground, with pBAC-T7-BCD7-VO1(I382L)-T7BCDx-CPRx. BCD stands forBiCistronic Design, and is described in Mutalik et. al. Nature Methods2013(10)4:354. Lower BCD numbers refer to higher translation rate. CPRsincluded SrCPR (SEQ ID NO: 62), SrCPR3 (SEQ ID NO: 80), AaCPR (SEQ IDNO: 68), PgCPR (SEQ ID NO: 82), AtCPR2 (SEQ ID NO: 72), AtCPR1 (SEQ IDNO: 70), eSrCPR1 (SEQ ID NO: 76), and eATR2 (SEQ ID NO: 74). Strainswere tested as in Example 5, at 30° C.

As shown in FIG. 20, SrCPR3, which was obtained through RNA sequencingstudies, exhibited a 1.3-fold improvement in oxygenated titer.

The CPR orthologs were retested at 34° C. The results are shown in FIG.20. Both SrCPR3 (SEQ ID NO: 80) and AaCPR (SEQ ID NO: 68) exhibited a1.3-fold improvement in oxygenated titer, even at the highertemperature. Oxygenated titers are comparable to those obtained at 30°C.

Example 9: Alcohol Dehydrogenase Enzymes to Alter Product Profile

The ability of alcohol dehydrogenases to convert nootkatols tonootkatone was evaluated. The following ADH enzymes were evaluated:

TABLE 15 CPR enzymes Gene UniProtID Organism reCDH Q9RA05 Rhodococcuserythropolis csDH1 A0A067H4B8 Citrus sinensis csDH2 A0A067H4S0 Citrussinensis csDH3 Citrus sinensis vvDH F6GX78 Vitis vinifera voDH1 csABA2A0A067DRA0 Citrus sinensis csDH A0A0A0KNF1 Cucumis sativus bdDH I1GLS4Brachypodium distachyon zzSDR F1SWA0 Zingiber zerumbet

Strains were evaluated as in Example 5, using MB2490 as the backgroundstrain (MP6-MEP FAB46-ScFPPS-L-VS1 MP6-VO1-o-SrCPR+p5-T7-BCD14-ADH).Briefly, MP6, Fab46 and T7 refer to the promoter for the attached geneor operon. Here MEP is an operon overexpressing E. coli dxs, idi, andispDF genes. The L between ScFPPS and VS1 refers to a short polypeptidelinker encoding (GSTGS) while -o- between VO1 and SrCPR refers to anoperonic construction in which an RBS sequence is inserted between thetwo genes. The plus denotes a plasmid following which is described as ap5 (five copy) plasmid with a promoter, BCD (described above) and theADH in question.

Four orthologs were identified (vvDH, csABA2, bdDH, and zzSDR) thatconvert (3-nootkatol to nootkatone, resulting in more than a 3-foldincrease in nootkatone titers. FIG. 21.

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1. A product comprising: an oxygenated terpene formulation comprising:nootkatone in an amount ranging from about 55% to about 60% (w/w);α-nootkatol in an amount ranging from about 20% to about 25% (w/w); andβ-nootkatol in an amount ranging from about 5% to about 10% (w/w),wherein the product is a flavor product, a natural flavor product, afragrance product, a cosmetic product, a cleaning product, a detergentproduct, a soap product, or a pest control product.
 2. The product ofclaim 1, wherein the flavor product is a natural flavor, beverage, achewing gum, a candy, an artificial flavor, or a flavor additive.
 3. Theproduct of claim 1, wherein the formulation further comprises valencene.4. The product of claim 3, wherein the valencene is present in theoxygenated terpene formulation in an amount ranging from about 5% toabout 10% (w/w).
 5. The product of claim 1, wherein at least one of thenootkatone, the α-nootkatol, and the β-nootkatol, is produced using abiosynthetic process.
 6. The product of claim 1, wherein the nootkatone,the α-nootkatol, and the β-nootkatol, are produced using at least onebiosynthetic process.
 7. A method of making a product for providingoxygenated terpenes, comprising: obtaining a formulation, saidformulation comprising: nootkatone in an amount ranging from about 55%to about 60% (w/w); α-nootkatol in an amount ranging from about 20% toabout 25% (w/w); and β-nootkatol in an amount ranging from about 5% toabout 10% (w/w), and incorporating the formulation into a consumerproduct, an industrial product, a flavor product, a fragrance product, acosmetic product, a cleaning product, a detergent product, a soapproduct, or a pest control product.
 8. The method of claim 7, whereinthe flavor product is a beverage, a chewing gum, a candy, a naturalflavor, an artificial flavor, or a flavor additive.
 9. The method ofclaim 7, wherein obtaining the formulation comprises contactingvalencene with Stevia rebaudiana Kaurene Oxidase (SrKO) or an SrKOderivative comprising a sequence having at least 70% sequence identityto SEQ ID NO: 37, SEQ ID NO: 38, or SEQ ID NO: 55, and having valenceneoxidizing activity in an in vivo or in vitro system to yield anoxygenated product.
 10. The method of claim 7, further comprisingprocessing the oxygenated product by fractional distillation to yieldtwo or more fractions of the oxygenated product.
 11. The method of claim10, further comprising blending the two or more fractions of theoxygenated product.
 12. The method of claim 7, further comprising addingan unoxygenated product to the oxygenated product, one or more fractionsof the oxygenated product, or the formulation.
 13. The method of claim12, wherein the unoxygenated product comprises valencene.
 14. The methodof claim 13, wherein the valencene is present in the formulation in anamount ranging from about 5% to about 10% (w/w).
 15. The method of claim7, wherein the formulation further comprises one or morenon-sesquiterpene components or sesquiterpene components.
 16. The methodof claim 15, wherein the one or more non-sesquiterpene components orsesquiterpene components is selected from Table 8A, Table 8B, and Table9.